Cross-protective human monoclonal antibody compositions

ABSTRACT

Cell lines have been produced that secrete human monoclonal antibodies capable of binding to molecules of different bacterial species. These antibodies have been found to be protective against lethal challenges of various bacterial genera. Pharmaceutical compositions containing these antibodies, which can be in combination with other monoclonal antibodies, blood plasma fractions and antimicrobial agents, and the prophylactic and therapeutic use of such compositions in the management of infections are included. Prior to filing of this patent application the continuous transformed human cell lines 9B10, 4F10, 4B9, 7D7, and 9C3, described herein, were deposited in the American Type Culture Collection and given the designations CRL 9006, CRL 9007, CRL 9008, CRL 9009, and CRL 9239, respectively.

This is a continuation of Ser. No. 08/058,987, filed May 5, 1993, nowabandoned, which is a continuation of Ser. No. 07/785,184, filed Oct.31, 1991, now abandoned, which is a continuation of Ser. No. 06/944,495,filed Dec. 19, 1986, now abandoned, which is a continuation-in-part ofSer. No. 06/828,005, filed Feb. 7, 1986, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the application of immunologicaltechniques to provide novel materials useful in treating and diagnosingbacterial infections and, more particularly, to the production andapplication of human monoclonal antibodies that are capable ofprotecting against infections caused by different genera of bacteria.

BACKGROUND OF THE INVENTION

Gram-positive and gram-negative bacteria may cause life-threateningdisease in infected patients. These bacterial infections often causesignificant morbidity and mortality. There is an increased incidence ofsuch infections in prematurely born infants, elderly patients, andpatients who have serious underlying medical conditions such as burns,surgical trauma, slow-healing wounds, or malignancies. These infectionsare typically of nosocomial origin (i.e., hospital-acquired), and occurparticularly in patients who have sustained prolonged hospitalizationassociated with surgical intervention, intravascular insult, orlong-term therapy with immunosuppressive agents or antibiotics. Inaddition, newborns who have an immature immune system are apparentlyacutely susceptible to neonatal sepsis and meningitis caused byparticular gram-negative and gram-positive bacteria.

Included among the most frequently encountered organisms ingram-negative and gram-positive disease are Escherichia coli (E. coli),Klebsiella pneumoniae (K. pneumoniae), Serratia marcescens (S.marcescens), Enterobacter aerogenes and cloacae (E. aerogenes/cloacae),Pseudomonas aeruginosa (P. aeruginosa), Neisseria meningitidis (N.meningitidis), Group B Streptococcus and Staphylococcus aureus (S.aureus) (Sonnenwirth, A. C., "The Enteric Bacilli and SimilarGram-Negative Bacteria," pp. 753-790, in Micro-biology, 2nd Edition,Davis, B. D., Dulbecco, R., Eisen, H. N., Ginserberg, H. S., Wood, W.B., and McCarty, M., Eds., Harper and Row, (1973); McCabe, W. R..,"Gram-Negative Bacteremia," Adv. Intern. Med., 19:135-138 (1974);Kreger, et al., "Gram-Negative Bacteremia III. Reassessment of Etiology,Epidemiology, and Ecology in 612 Patients," Am. J. Med. 68:332-343(1980); Robbins, J. B., et al., "Escherichia coli K1 CapsularPolysaccharide Associated With Neonatal Meningitis," New Engl J. Med.,290:1216-1220 (1974); and Hughs, J. M., et al., "Nosocomial InfectionSurveillance, 1980-1982," Morb. Mort. Weekly Report, 32:1SS-16SS(1983)). Of these infections, usually several, but not all, serotypes ofcertain gram-negative bacteria, e.g., E. coli, K. pneumoniae, E.aerogenes/cloacae, P. aeruginosa, and S. marcescens, cause bacteremiaamong the adult population. In contrast to adults, the immunologicallyimmature neonate is particularly susceptible to septicemia andmeningitis caused by the encapsulated strains of E. coli, N.meningitidis Group B, Hemophilus influenzae type B, and the five typestrains of Group B Streptococcus. Although other bacteria may also causethese infections, the bacteria cited above are the predominant isolatesfrom the aforementioned blood infections.

Antibiotics have long been the primary therapeutic tool for the controland eradication of gram-positive and gram-negative infections. However,the continued incidence and severity of the infections, the continualemergence of antibiotic resistant bacterial strains, and the inherenttoxicity of some antibiotics, point to the limitations of antibiotictherapy. These observations have prompted the search for alternativeprophylactic and therapeutic approaches.

It is widely believed that antibodies reactive with structuresaccessible (externally exposed) on live bacteria may facilitatebacterial destruction by any of several mechanisms. Included among thesemechanisms are: (1) direct lysis of the bacteria in the presence ofserum complement, (2) bacteriostasis, by the blockading of nutrientscavenger receptors, (3) opsonization and subsequent phagocytosis of thebacteria in the presence or absence of serum complement, or (4)prevention of attachment of the bacteria to host tissues (Mims, C. A.,"Recovery from Infection," in The Pathogenesis of Infectious Disease,pp. 198-222, Mims, C. A., Ed., Academic Press (1982)). For bacteria thatpossess surface carbohydrate molecules, such as lipopolysaccharide (LPS)and/or capsules, antibody appears to be most effective via opsonizationmechanisms (Kaijser, B., et al., "The Protective Effect Against E. coliof O and K Antibodies of Different Immunoglobulin Classes," Scand. J.Immunol., 1:276 (1972)). Therefore, antibodies directed to theseaccessible carbohydrate structures may provide an effective means forbacterial elimination.

In general, mammals that are exposed to disease-producing bacteriaproduce antibodies that are specific for LPS or capsule. These antigensare chemically diverse structures composed of frequently repeatingoligosaccharide molecules and whose presence determines the serotype ofbacterial strains. Since they are often the immunodominant bacterialantigens, serotype specific antibodies (anti-LPS or capsule) have beenthe most studied of potentially therapeutic antibodies. However, becauseof the limited cross-reactivity of these antibodies, and the apparenthighly diverse nature of carbohydrate antigens on pathogenicgram-positive and gram-negative bacteria, it would be extremelydifficult and costly to produce a therapeutic formulation containingonly serotype specific antibodies (see, e.g., Kaijser, B. and Ahlstedt,S., "Protective Capacity of Antibodies Against Escherichia coli O and KAntigens, " Infect. Immun., 17:286-292 (1977); and Morrison, D. C. andRyan, J. L., "Bacterial Endotoxins and Host Immune Response, "Adv.Immunol , 28:293-450 (1979)). Regardless, various reports havestimulated visions that immunotherapeutic approaches could be found totreat gram-negative bacterial disease.

Fractionated human plasma, enriched for immune globulins containingspecific and protective antibodies against the infecting organisms, havebeen somewhat effective against P. aeruginosa infections. (Collins, M.S. and Robey, R. E., "Protective Activity of an Intravenous ImmuneGlobulin (Human) Enriched in Antibody Against LipopolysaccharideAntigens of Pseudomonas aeruginosa, " Amer. J Med., 3:168-174 (1984)).However, commercial products are not yet readily available due tocertain inherent limitations which have prevented their widespread usein the treatment of life-threatening bacterial disease.

One such limitation associated with immune globulin compositions is thatthey are assembled from large pools of plasma samples that have beenpreselected for the presence of a limited number of particularantibodies. Typically, these pools consist of samples from a thousanddonors who may have low titers to some pathogenic bacteria. Thus, atbest, there is only a modest increase in the resultant titer of desiredantibodies.

Another such limitation is that the preselection process itself requiresvery expensive, continuous screening of the donor population to assureproduct consistency. Despite considerable effort, product lots can stillvary between batches and geographic regions.

Yet another such limitation inherent in immune globulin compositions isthat their use results in coincident administration of large quantitiesof extraneous proteinaceous substances (e.g., viruses) having thepotential to cause adverse biologic effects. The combination of lowtiters of desired antibodies and high content of extraneous substancesoften limits, to suboptimal levels, the amount of specific and thusbeneficial immune globulin(s) administrable to the patient.

In 1975, Kohler and Milstein reported that certain mouse cell linescould be fused with mouse spleen cells to create hybridomas which wouldsecrete pure "monoclonal" antibodies (Kohler, G. and Milstein, C.,"Continuous Cultures of Fused Cells Secreting Anti-body of PredefinedSpecificity," Nature, 256:495-497 (1975)). With the advent of thistechnology, the potential existed to produce murine antibodies to anyparticular determinant or determinants on antigens.

Using this technology, mouse monoclonal antibodies have been derivedfrom mice immunized with polysaccharide from Neisseria meningitidisGroup B. These murine IgM monoclonal antibodies were observed to bindand opsonize several K1-positive E. coli strains regardless of their LPSserotypes (Cross, "Evaluation of Immunotherapeutic Approaches for thePotential Treatment of Infections Caused by K2-Positive Escherichiacoli," J. Infect. Dis. 147:68-76 (1983), Soderstrom, "Serologial andFunctional Properties of Monoclonal Antibodies to Escherichia coli TypeI Pilus and Capsular Antigens," Prog. Allergy 33:259-274 (1983), andCross, A. S., et al., "The Importance of the K1 Capsule in InvasiveInfections Caused by Escherichia coli," J. Inf. Dis., 149:184-193(1984)). Moreover, the monoclonal antibodies were protective in miceagainst lethal challenges with E. coli K1 and Group B meningococcalorganisms (Cross, J. Infect. Dis. 147:68-76 (1983), and Soderstrom,supra). In another example, mouse monoclonal antibodies specific to typeIII Group B Streptococcus were reported to be protective in a mouseexperimental infection model (Egan, M. L., et al., "Protection of Micefrom Experimental Infection with Type III Group B Streptococcus UsingMonoclonal Antibodies," J. Exp. Med., 1:1006-1011 (1983)).

A mouse monoclonal antibody, while useful in treating mice, has majordisadvantages for use in humans. The human immune system is capable ofrecognizing any mouse monoclonal antibody as a foreign protein. This canresult in accelerated clearance of the antibody and thus abrogation ofits pharmacological effect (Levy, R. and Miller, R. A., "Tumor Therapywith Monoclonal Antibodies," Fed. Proc., 42:2650-2656 (1983)). Moreseriously, this could conceivably lead to shock and even death fromallergic reactions analogous to "serum sickness." Clinical experiencehas shown that anti-mouse immunoglobulin responses have limited theutility of these antibodies in approximately one-half of the patientsreceiving mouse monoclonal antibodies for treatment of various tumors(Sears, H. F., et al., "Phase I Clinical Trial of Monoclonal Antibody inTreatment of Gastrointestinal Tumor," Lancet, 1:762-764 (1982); andMiller, R. A., et al., "Monoclonal Antibody Therapeutic Trials in SevenPatients with T-Cell Lymphoma," Blood, 62:988-995 (1983)).

Accordingly, there is a need for human monoclonal antibodies which areprotective against gram-negative and gram-positive bacterial disease.However, the diverse antigenicity of gram-positive and gram-negativedisease-causing bacteria strongly suggests that producing serotypespecific human monoclonal antibodies to each of the many importantbacterial pathogens would be impractical.

The diverse antigenicity of gram-negative bacteria is attributed to thevariable regions of the lipopolysaccharide (LPS), a molecule associatedwith the outer membrane of gram-negative organisms. The LPS molecule isgenerally considered to be composed of three structural regions. Theregion closest to the outer membrane is the so-called lipid A portion ofLPS. This structurally conserved region possesses the endotoxic activityassociated with gram-negative disease. The second structural region,termed core, is linked to a lipid A often via a2-keto-3-deoxy-D-mannooctonate residue (KDO) and, similar to the lipid Aregion, is not usually accessible to antibody when the third outermostregion of LPS is present. Although this region is partially conservedwithin some gram-negative bacterial species, many deviations in completecore have been found among members of the family Enterobacteriaceae. Theoutermost region of an LPS molecule is composed of repeatingoligosaccharide units and is known as the O-specific side chain. Thesugars in these oligosaccharide units comprise molecular entities thatexhibit serotype specific structural antigenic diversity. Thus, thesugars themselves, their sequence, and their linkages determine O-sidechain antigenicity via their tertiary structure. Antibodies to theseO-groups have generally been found to be serotype specific. Serotypesare typically defined by their reactivity with monospecific antisera,which possess binding activity for only one particular antigenicdeterminant. See generally, Mayer et al., Meths. Microbiology,18:157-201 (1985).

Antisera to the core and lipid A regions of LPS have been produced inefforts to demonstrate protection against gram-negative infection.Sakulramrung and Domingue, J. Inf. Dis., 151:995-1104 (1985); McCabe, etal., J. Infect. Dis., 1365:516 (1977); and Mullan, et al., Infect.Immun., 10:1195-1201 (1974). More recently, mouse and human monoclonalantibodies reactive with the conserved regions have been produced.Although these antibodies have sometimes shown partial in vivo efficacyin tailored model systems (Teng, et al., Proc. Natl. Acad. Sci. U.S.A.82:1790 (1985); and Bogard and Kung, Patent Application No. WO85/01659),other laboratories have not been able to demonstrate similar effects.See Elkins and Metcalf, Infect. Immun. 48:597 (1985); and Gigliotti andShenap, J. Inf. Dis. 151:1005-1011 (1985). Moreover, these antibodiesgenerally do not react with (bind to) intact, viable gram-negativebacteria or to purified LPS molecules. These findings suggest that it isdoubtful the core or lipid A portions of LPS on bacteria in theirnatural and infectious state would be accessible to antibody. It is alsowell accepted that anti-core or anti-lipid A antibodies will not reactwith gram-positive bacteria because the latter do not possess LPS. Inview of these findings, it is unlikely that monoclonal antibodies to theconserved core or lipid A regions of LPS will be efficacious in thetreatment of human gram-negative or, for that matter, gram-positivebacterial disease.

Thus, there still exists a significant need for human monoclonalantibodies that are broadly (intergenus) cross-protective againstgram-positive and gram-negative bacterial diseases, as well as formethods for practical production and use of such antibodies. The presentinvention fulfills these needs.

SUMMARY OF THE INVENTION

Novel cell lines are provided which produce human monoclonal antibodiescapable of specifically cross-reacting with a plurality of bacterialspecies by binding an accessible epitope comprising a non-corecarbohydrate moiety present on at least two different bacterial species.Additionally, methods are provided for prophylactically treating a humanpatient susceptible to bacterial infection and therapeutically treatinga patient suffering from such an infection by administering an effectiveamount of a composition comprising a plurality of human monoclonalantibodies, wherein at least one of these antibodies is capable ofreacting with a non-core carbohydrate antigenic determinant shared bytwo or more bacterial species. The composition preferably includes aphysiologically acceptable carrier, and may also contain any one or moreof the following: additional human monoclonal antibodies capable ofreacting with other bacterial genera; a gammaglobulin fraction fromhuman blood plasma; a gammaglobulin fraction from human blood plasma,where the plasma is obtained from a human exhibiting elevated levels ofimmunoglobulins reactive with one or more bacterial genera; and one ormore antimicrobial agents.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the present invention, novel cells capable ofproducing human monoclonal antibodies and compositions comprising suchantibodies are provided, such compositions being capable of selectivelyreacting with a plurality of bacterial genera responsible fornosocomial, neonatal, or other infections, where individual antibodiestypically react with non-core carbohydrate epitopes present on multiplebacterial genera. The subject cells have identifiable chromosomes inwhich the germ-line DNA from them or a precursor cell has rearranged toencode an antibody or binding fragment thereof having a binding site foran antigenic determinant (epitope) shared by carbohydrate moleculesfound on at least some serotypes of two or more bacterial genera. Thesehuman monoclonal antibodies can be used in a wide variety of ways,including for diagnosis, prophylaxis and therapy of bacterial disease.

Typically, the cells of the present invention will be cells capable ofstable production of a human antibody in culture, particularlyimmortalized human lymphocytes that produce protective human monoclonalantibodies to non-core carbohydrate determinants on accessible moleculesshared by at least two bacterial species. By "accessible" is meant thatthe non-core carbohydrate determinants are physically available in theenvironment of use for direct interaction with the monoclonalantibodies. The monoclonal antibodies so provided are useful in thetreatment or prophylaxis of serious disease caused by a wide range ofbacterial infections. Furthermore, those non-core carbohydrate moleculesthat are released into the surrounding environment are also free tointeract directly with the antibody molecules and be cleared via thereticuloendothelial system.

The compositions containing the monoclonal antibodies of the presentinvention will typically be useful in the therapeutic and prophylactictreatment of nosocomial, neonatal, and other infections. As nosocomialinfections are typically caused by infections from the followingbacteria: Escherichia coli, Pseudomonas aeruginosa, Klebsiellapneumoniae, Enterobacter aerogenes/cloacae, Serratia marcescens, andStreptococcus agalactiae Group B, antibody compositions protectiveagainst two, three, four, or more of such bacteria would be preferred.Similarly, for neonatal use, such as in neonatal sepsis and meningitis,the antibodies are desirably specific for two or more of the followingbacterial organisms: Escherichia coli K1, Neisseria meningitidis GroupB, Streptococcus agalactiae Group B, and Hemophilus influenzae type B.Other common infectious bacteria include: Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus pneumoniae, Proteus mirabilis,Proteus vulgaris, Bacteroides fragilis, Pseudomonas cepacia,Mycobacterium tuberculosis, Providencia morganii, Salmonella typhi,Pneumocystis carinii, Acinetobacter herellea, Pasturella multocida,Klebsiella oxytoca. For additional relevant pathogenic bacteria known tothose skilled in the art, see, Hughs, J. M., et al., "NosocomialInfection Surveillance, 1980-1982,"Morb. Mort. Weekly Report,32:1SS-16SS (1983), and, generally, Microbiology, 3rd Edition, Davis, B.D., Dulbecco, R., Eisen, H. N., Ginserberg, H. S., Wood, W. B., andMcCarty, M., Eds., Harper and Row (1980), both of which are incorporatedherein by reference. The monoclonal antibodies will react withindividual members or all of the members of a particular bacterialspecies, where the members may be distinguished by their surfaceepitopes, particularly LPS or capsule sites, e.g. serotypes.

The unexpected discovery of monoclonal antibody cross-reactivity acrossvarious bacterial species, including the clinically important specieslisted above, provides novel means for therapeutic and prophylactictreatments. By utilizing pre-selected cross-reactive antibodies incombination, a mixture of a few antibodies can be produced for treatmentagainst a number of different species of infectious bacteria.

By way of example, and not of limitation, a mixture of two monoclonalantibodies, one cross-reactive with at least two bacterial species ofclinical significance and the second cross-reactive with at least two orthree different species, will be useful in treatment against four, five,six or more different species. Adding a third or fourth monoclonalantibody, each one of which is cross-reactive with at least twoclinically important species--even if one or more of the species is thesame as that recognized by the first and/or second antibody, willincrease usefulness in treatment against five to ten or more species. Ofcourse, it may be necessary to also add one or more monoclonalantibodies, each specific for just a single pre-selected bacterialspecies, for example, when monoclonal antibodies cross-reactive withthat species are unavailable.

Similarly, new methods of treating bacterial infections are alsoprovided based on the discovery. Again, by way of example and notlimitation, one novel method entails treating a patient suspected ofhaving or being susceptible to a bacterial infection caused by aselected bacterial species. The treatment includes administering acomposition comprising a monoclonal antibody reactive with the bacterialspecies suspected of causing the infection, wherein the monoclonalantibody was initially characterized as reactive with a differentbacterial species.

Another example is a method of treating bacterial infections byadministering compositions comprising a plurality of monoclonalantibodies reactive with a substantial proportion (i.e., greater than50%, preferably 60% to 80% or more, most preferably about 90%) ofpre-selected, clinically important bacterial species, wherein the numberof antibodies is at least about two less than the number of bacterialspecies. Typically, if "n" represents the number of bacterial species,the composition will comprise about n-2 antibodies, more typically aboutn-4 to n-8 or less antibodies for treatment against up to about 15 to 20bacterial species. In situations where treatment against a broadspectrum (e.g., 25 to 50 or more) of bacterial species is desired, thecomposition will typically comprise n-10 to n-20 antibodies or less.

Preparation of monoclonal antibodies can be accomplished byimmortalizing the expression of nucleic acid sequences that encode forantibodies or binding fragments thereof specific for a non-corecarbohydrate epitope present on multiple bacterial species. Typically,the monoclonal antibodies are produced by cell-driven Epstein-Barr Virus(EBV) transformation of lymphocytes obtained from human donors who are,or have been exposed to the respective gram-negative bacteria. Theantibody-secreting cell lines so produced are characterized ascontinuously growing lymphoblastoid cells that possess a diploidkaryotype, are Epstein-Barr nuclear antigen (EBNA) positive, and secretemonoclonal antibody of either IgG, IgM, IgA, or IgD isotype. Thecell-driven transformation process itself is an invention assigned toGenetic Systems Corporation and is described in detail in U.S. Pat. No.4,464,465 which is incorporated herein by reference. The monoclonalantibodies may be used intact, or as fragments, such as F_(v), Fab,F(ab')₂, but usually intact.

Alternatively, cell lines producing the antibodies could be produced bycell fusion between suitably drug-marked human myeloma, mouse myeloma,or human lymphoblastoid cells with human B-lymphocytes to yield hybridcell lines.

The cell lines of the present invention may find use other than for thedirect production of the human monoclonal antibodies. The cell lines maybe fused with other cells (such as suitably drug-marked human myeloma,mouse myeloma, or human lymphoblastoid cells), to produce hybridomas,and thus provide for the transfer of the genes encoding the humanmonoclonal antibodies. Alternatively, the cell lines may be used as asource of the DNA encoding the immunoglobulins, which may be isolatedand transferred to cells by techniques other than fusion. In addition,the genes encoding the monoclonal antibodies may be isolated and used inaccordance with recombinant DNA techniques for the production of thespecific immunoglobulin in a variety of hosts. Particularly, bypreparing cDNA libraries from messenger RNA, a single cDNA clone, codingfor the immunoglobulin and free of introns, may be isolated and placedinto suitable prokaryotic or eukaryotic expression vectors andsubsequently transformed into a host for ultimate bulk production.

The lymphoblastoid or hybrid cell lines may be cloned and screened inaccordance with conventional techniques, with the antibodies that arecapable of binding to the epitopes of different bacterial generadetected in the cell supernatants.

The monoclonal antibodies of this invention find particular utility ascomponents of pharmaceutical compositions containing a therapeutic orprophylactic amount of at least one of the monoclonal antibodies of thisinvention in conjunction with a pharmaceutically effective carrier. Apharmaceutical carrier can be any compatible, non-toxic substancesuitable for delivery of the monoclonal antibodies to the patient.Sterile water, alcohol, fats, waxes, and inert solids may be included inthe carrier. Pharmaceutically accepted adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. Such compositions can contain a single monoclonal antibodycross-reactive with non-core carbohydrate epitopes shared by two or morebacterial species that cause, for example, nosocomial and neonatal(e.g., sepsis or meningitis) infections. Alternatively, a pharmaceuticalcomposition can contain two or more monoclonal antibodies to form a"cocktail." For example, a cocktail containing human monoclonalantibodies each protective against two or more gram-negative bacterialgenera responsible for human infections, would have activity against thegreat majority of the common clinical isolates. If desired, one or moreof the monoclonal antibodies could be selected to be cross-reactive withgram-positive bacteria as well, making even broader product applicationsfeasible.

Of interest are prophylactic and/or therapeutic monoclonal antibodycompositions capable of reacting with non-core carbohydrate determinantsshared by three or more, usually at least five, and more usually atleast ten, and up to fifteen or more bacterial serotypes, which includesat least two, usually at least three, more usually at least five, andusually fewer than ten bacterial genera.

Of particular interest are monoclonal antibody compositions which reactwith at least about three, preferably at least about five and up to andincluding all of the following common nosocomial infection-causingbacteria: Escherichia coli, Pseudomonas aeruginosa, Klebsiellapneumoniae, Enterobacter aerugenes/cloacae, Serratia marcescens, andStreptococcus agalactiae Group B. For treatment of neonatal infections,desirably the compositions will react with at least two, usually atleast three, and more usually at least four and up to and including allof the following infection-causing bacterial genera: Escherichia coliK1, Neisseria meningitidis Group B, Streptococcus agalactiae Group B,Hemophilus influenzae type B, Staphylococcus aureus, and Staphylococcusepidermidis.

Each of the compositions will include at least two, usually at leastthree to five, and more usually six to ten human monoclonal antibodies,where at least one antibody reacts with non-core carbohydrate epitopes(e.g., of the LPS molecules) shared by two or more bacterial genera andproviding protection. Typically, the antibody will not bind to allserotypes of each bacterium, but may bind to two, three or moreserotypes. Desirably, there will be at least one monoclonal antibodywhich binds to an accessible non-core carbohydrate moiety of at leasttwo genera of gram-negative bacteria and at least one monoclonalantibody that binds to an accessible carbohydrate moiety of agram-negative bacterium and a gram-positive bacterium.

The mole ratio of the various monoclonal antibody components willusually not differ one from the other by more than a factor of 10, moreusually by not more than a factor of 5, and will usually be in a moleratio of about 1:1-2 to each of the other antibody components.

The human monoclonal antibodies may also find use individually,particularly where the pathogen has been identified or is limited to anarrow range of pathogens within the binding spectrum of the particularantibody.

The human monoclonal antibodies of the present invention may also beused in combination with other monoclonal antibodies as described indetail on U.S. Pat. No. 5,378,812 as well as existing blood plasmaproducts, such as commercially available gamma globulin and immuneglobulin products used in prophylactic or therapeutic treatment ofbacterial disease in humans. Preferably, for immune globulins the plasmawill be obtained from human donors exhibiting elevated levels ofimmunoglobulins reactive with various infectious bacterial genera. Seegenerally, the compendium "Intravenous Immune Globulin and theCompromised Host," Amer. J. Med., 76(3a), Mar. 30, 1984, pgs 1-231,which is incorporated herein by reference.

The monoclonal antibodies of the present invention can be used asseparately administered compositions given in conjunction withantibiotics or antimicrobial agents. Typically, the antimicrobial agentsmay include a penicillin or cephalosporin (e.g., carbenicillin,penicillin G, or the like) in conjunction with an aminoglycoside (e.g.,gentamicin, tobramycin, etc.), but numerous additional agents (e.g.,cephalosporins, sulfa drugs, etc.) well-known to those skilled in theart may also be utilized.

The human monoclonal antibodies and pharmaceutical compositions thereofof this invention are particularly useful for oral or parenteraladministration. Preferably, the pharmaceutical compositions may beadministered parenterally, i.e., subcutaneously, intramuscularly orintravenously. Thus, this invention provides compositions for parenteraladministration which comprise a solution of the human monoclonalantibody or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.4% saline, 0.3% glycine and thelike. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, i.e.,from less than about 0.5%, usually at or at least about 1% to as much as15 or 20% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intramuscular injectioncould be made up to contain 1 ml sterile buffered water, and 50 mg ofmonoclonal antibody. A typical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 150mg of monoclonal antibody. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pa.(1980), which is incorporated herein by reference.

The monoclonal antibodies of this invention can be lyophilized forstorage and reconstituted in a suitable carrier prior to use. Thistechnique has been shown to be effective with conventional immuneglobulins and art-known lyophilization and reconstitution techniques canbe employed. It will be appreciated by those skilled in the art thatlyophilization and reconstitution can lead to varying degrees ofantibody activity loss (e.g., with conventional immune globulins, IgMantibodies tend to have greater activity loss than IgG antibodies) andthat use levels may have to be ad- justed to compensate.

The compositions containing the present human monoclonal antibodies or acocktail thereof can be administered for the prophylactic and/ortherapeutic treatment of bacterial infections. In therapeuticapplication, compositions are administered to a patient alreadyinfected, in an amount sufficient to cure or at least partially arrestthe infection and its complications. An amount adequate to accomplishthis is defined as a "therapeutically effective dose." Amounts effectivefor this use will depend upon the severity of the infection and thegeneral state of the patient's own immune system, but generally rangefrom about 1 to about 200 mg of antibody per kilogram of body weightwith dosages of from 5 to 25 mg per kilogram being more commonly used.It must be kept in mind that the materials of this invention maygenerally be employed in serious disease states, that islife-threatening or potentially life-threatening situations, especiallybacteremia and endotoxemia. In such cases, in view of the absence ofextraneous substances and the absence of "foreign substance" rejectionswhich are achieved by the present human monoclonal antibodies of thisinvention, it is possible and may be felt desirable by the treatingphysician to administer substantial excesses of these antibodies.

In prophylactic applications, compositions containing the presentantibody or a cocktail thereof are administered to a patient not alreadyinfected by the corresponding bacteria to enhance the patient'sresistance to such potential infection. Such an amount is defined to bea "prophylactically effective dose." In this use, the precise amountsagain depend upon the patient's state of health and general level ofimmunity, but generally range from 0.1 to 25 mg per kilogram, especially0.5 to 2.5 mg per kilogram.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the antibody(ies) of this invention sufficient toeffectively treat the patient.

Monoclonal antibodies of the present invention can further find a widevariety of utilities in vitro. By way of example, the monoclonalantibodies can be utilized for bacterial typing, for isolating specificbacterial strains or fragments thereof, for vaccine preparation, or thelike.

For diagnostic purposes, the monoclonal antibodies may either be labeledor unlabeled. Typically, diagnostic assays entail detecting theformation of a complex through the binding of the monoclonal antibody tothe LPS of the organism. When unlabeled, the antibodies find use inagglutination assays. In addition, unlabeled antibodies can be used incombination with other labeled antibodies (second antibodies) that arereactive with the monoclonal antibody, such as antibodies specific forhuman immunoglobulin. Alternatively, the monoclonal antibodies can bedirectly labeled. A wide variety of labels may be employed, such asradionuclides, fluorescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, ligands (particularly haptens), etc.Numerous types of immunoassays are available, and by way of example,some of the assays are described in U.S. Pat. Nos. 3,817,827; 3,850,752;3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876,all of which are incorporated herein by reference.

Commonly, the monoclonal antibodies of the present invention areutilized in enzyme immunoassays, where the subject antibodies, or secondantibodies from a different species, are conjugated to an enzyme. When asample, such as human blood or lysate thereof, containing one or morebacteria of a certain genus or serotype, is combined with the subjectantibodies, binding occurs between the antibodies and those moleculesexhibiting the selected epitopes. Such cells may then be separated fromthe unbound reagents, and a second antibody (labeled with an enzyme)added. Thereafter, the presence of the antibody-enzyme conjugatespecifically bound to the cells is determined. Other conventionaltechniques well known to those skilled in the art may also be utilized.

Kits can also be supplied for use with the subject antibodies in thedetection of bacterial infection or for the presence of a selectedantigen. Thus, the subject monoclonal antibody composition of thepresent invention may be provided, usually in a lyophilized form in acontainer, either alone or in conjunction with additional antibodiesspecific for other gram-negative bacteria. The antibodies, which may beconjugated to a label or unconjugated, are included in the kits withbuffers, such as Tris, phosphate, carbonate, etc., stabilizers,biocides, inert proteins, e.g., bovine serum albumin, or the like.Generally, these materials will be present in less than about 5% wt.based on the amount of active antibody, and usually present in totalamount of at least about 0.001% wt. based again on the antibodyconcentration. Frequently, it will be desirable to include an inertextender or excipient to dilute the active ingredients, where theexcipient may be present in from about 1 to 99% wt. of the totalcomposition. Where a second antibody capable of binding to themonoclonal antibody is employed, this will usually be present in aseparate vial. The second antibody is typically conjugated to a labeland formulated in an analogous manner with the antibody formulationsdescribed above.

The following experimental data and information are offered by way ofexample and not limitation.

EXAMPLE I

This EXAMPLE demonstrates methods for the production of a humanmonoclonal antibody that possesses intergenus cross-reactivity againstmembers of the genera Escherichia coli (E. coli), Serratia marcescens(S. marcescens), Klebsiella pneumoniae (K. pneumoniae), and Enterobacteraerogenes (E. aerogenes). Further, this EXAMPLE demonstrates the in vivoprotective activity of said antibody, against a lethal challenge ofhomologous (cross-reacting) E. coli, and E. aerogenes serotypes.

A. Obtaining Suitable Human Cells

Suitable human B cells (lymphocytes) were obtained from the peripheralblood of an individual known to have harbored the disease cysticfibrosis. Mononuclear cells were separated from the peripheral blood bystandard centrifugation techniques on Ficoll-Paque (Boyum, A.,"Isolation of Mononuclear Cells and Granulocytes From Human Blood,"Scand. J. Clin. Lab. Invest., 21. Suppl. 97:77-89, (1968)) and washedtwice in calcium-magnesium free phosphate buffered saline (PBS) prior tosuspension in 1 ml of 90% fetal bovine serum (FBS) and 10% dimethylsulfoxide and freezing at -196° C. in liquid nitrogen.

When the mononuclear cells were to be transformed, an ampule containing5×10⁷ cells was rapidly thawed at 37° C. The cell suspension was addedto 10 ml of Iscove's medium containing 15% FBS and centrifuged at roomtemperature for 10 min at 250×g. The mononuclear cells were depleted ofT cells (lymphocytes) using a modified E-rosetting procedure. Briefly,the cells were resuspended to a concentration of 1×10⁷ cells/ml in PBScontaining 20% FBS at 4° C. One ml of this suspension was placed in a17×100 mm polystyrene round bottom tube to which was added 1×10⁹2-amino-ethyl-isothiouronium bromide (AET)-treated sheep red blood cellsfrom a 10% (v/v) solution in Iscove's medium (Madsen, M. and Johnson, H.E., "A Methodological Study of E-rosette Formation Using AET TreatedSheep Red Blood Cells," J. Immun. Methods, 27:61-74, (1979)). Thesuspension was vigorously mixed for 5-10 min at 4° C. and the E-rosettedcells were removed by centrifugation through Ficoll-Paque for 8 min at2500×g at 4° C. E-rosette negative peripheral blood mononuclear cells(E⁻ PMBC) banding at the interface were washed once in Iscove's mediumand resuspended in same containing 15% w/v FBS (g/ml) , L-glutamine (2mM/1) , sodium pyruvate (1 mM/1), penicillin 100 IU/ml), streptomycin(100 μg/ml), hypoxanthine (1×10⁻⁴ M), aminopterin (4×10⁻⁷ M) andthymidine (1.6×10⁻⁷ M). This medium is hereafter referred to as HATmedium.

B. Cell-Driven Transformation of Peripheral BLood Mononuclear Cells

Cell-driven transformation of the E⁻ PBMC was accomplished bycocultivating the E⁻ PBMC with a transforming cell line. Thetransforming cell line was an EBNA positive human lymphoblastoid cellline derived by ethylmethane sulphonate mutagenesis of the GM 1500lymphoblastoid cell line. Selection in the presence of 30 μg/ml6-thioguanine rendered the cells hypoxanthineguanine phosphoribosyltransferase deficient and thus HAT sensitive. The cell line isdenominated the 1A2 cell line and was deposited at the American TypeCulture Collection (A.T.C.C.) on Mar. 29, 1982 under A.T.C.C. No. CRL8119. 1A2 cells in logarithmic growth phase were suspended in HAT mediumand combined with the E⁻ PBMC at a ratio of 30 1A2 cell per E⁻ PBMC. Thecell mixture was plated into 10 round-bottom 96-well microtiter platesat a concentration of 62,000 cells/well in a volume of 200 μl/well, andthe culture incubated at 37° C. in a humidified atmosphere containing 6%CO₂. Cultures were fed five days post transformation by replacement ofhalf the supernatant with fresh HAT medium. The wells were observedevery other day on an inverted microscope for signs of cellproliferation. Ten days after plating the cell mixture and after the 1A2cells had died due to HAT selection, feeding of the the wells wasaccomplished with a new media formulation identical to HAT media exceptthat it lacked the aminopterin component. Fifteen days post plating, itwas observed that 100% of the wells contained proliferating cells andthat in most of the wells, the cells were of sufficient density forremoval and testing of supernatants for anti-E. coli or anti-S.marcescens antibody.

C. Detection of Specific Antibody Secreting Cells

Supernatants were screened for the presence of anti-E. coli or anti-S.marcescens antibodies using an enzyme-linked immunosorbent assay (ELISA)technique (Engvall, E., "Quantitative Enzyme Immunoassay (ELISA) inMicrobiology," Med. Biol., 55:193-200, (1977)). The antigen platesconsisted of a series of flat-bottom 96-well Immunlon 2 microtiterplates, the wells of which contained a mixture of either viable E. colior S. marcescens serotypes affixed to the well surfaces withpoly-L-lysine (PLL). Briefly, 50 μl of PLL (1 μg/ml) in PBS was added toeach well for 30 min at room temperature (RT). The plates were washedthree times with PBS and either PBS or 50 μl of a mixed bacteriasuspension at O.D.₆₆₀ =0.2 was added to each well. The plates wereincubated at 37° C. for 60 min and washed 3 times with saline/0.02%Tween 20 (saline/T) to removed unattached bacteria. Various antigenplates used in the screen included: 1) a mixture of E. coli serotypes 01(A.T.C.C. No. 23499) and 04 (A.T.C.C. No. 12791); 2) a mixture of S.marcescens serotypes 07, 015, 016 and 018 (all reference typing strainswere obtained from the Communicable Disease Center (CDC) Atlanta, Ga.);and 3) a microtiter plate with no bacteria.

For the ELISA procedure, assay wells were first blocked with 200 μl of amixture containing 5% w/v dry non-fat milk, 0.0001% Anti-Foam A, and0.01% w/v Thimerosal in 500 ml PBS to prevent non-specific proteinbinding. After incubation for 1 hour at RT, the plates were washed threetimes with 200 μl/well/wash of saline/T. To each well was added 50 μl ofa mixture containing 0.1% Tween 20 and 0.2% bovine serum albumin in PBS(PTB). Supernatants from wells of the culture plate were replica platedinto corresponding wells of the antigen and control plates (50 μl/well)and the plates were incubated at RT for 30 min. The supernatants werethen removed, plates were washed five times with saline/T, and 50 μl ofbiotinylated goat anti-human immunoglobulin (Ig)(TAGO #9303040 diluted1:250 in PTB) was added to each well. After a 30 min incubation at RTthe biotinylated reagent was removed, the wells washed five times withsaline/T and 50 μl of a preformed avidin:biotinylated horseradishperoxidase complex (Vectastain ABC Kit, Vector Laboratories) was addedto each well. After 30 min. at RT the Vectastain ABC reagent wasremoved, the wells were washed five times with saline/T, and 100 μl ofsubstrate (0.8 mg/ml ortho-phenylenediamine dihydrochloride in 100 mMcitrate buffer, pH 5.0 plus 0.03% H₂ O₂ in deionized H₂ O mixed in equalvolumes just before plating) added to each well. After 30 min incubationin the dark, 50 μl of 3N H₂ SO₄ was added to each well to terminate thereaction. Culture supernatants which contained antibody reactive withthe bacteria coated plates were detected by measuring absorbance at 490nm on a Dynatech MR 580 microELISA reader.

Culture supernatants from six transformations were analyzed by the abovemethod resulting in the identification of one well (7D7) which possessedactivity on the E. coli and S. marcescens serotype plates, but not onthe control plates. It was determined in subsequent ELISA's withindividual E. coli serotypes, that this well contained antibody reactivewith at least, the E. coli serotypes: 08, (A.T.C.C. No. 23504), and 075(A.T.C.C. No. 12798), but not 04, 06:K2, 08:K8, 09:K9 or 022:K13(A.T.C.C. Nos. 12791, 19138, 23501, 23505 and 23517, respectively).Further, this well contained antibody reactive with the S. marcescensserotypes 012, 013 and 015, but not any other of the twenty known S.marcescens LPS serotypes.

D. Cloning of Specific Antibody Producing Cells

The cells in well 7D7 were subjected to several rounds of cloning (four)until all clonal supernatants assayed by the above ELISA procedure gavea positive reaction on E. coli serotypes 08 and 075 and on S. marcescensserotypes 012, 013 and 015. There was never an example when any clonalsupernatant demonstrated segregation in its reactivity pattern,suggesting that the culture supernatant from well 7D7 possessed trueintergenus cross-reactivity to the E. coli and S. marcescens serotypesset forth and did not contain more than one cell line (eachdemonstrating individual serotype reactivity). Cells were cloned bylimiting dilution in round-bottom 96-well plates in the absence offeeder cells. Media consisted of Iscove's medium containing 15% v/v FBS,L-glutamine (2 mM/l ), sodium pyruvate (1 mM/1), penicillin (100 IU/ml),and streptomycin (100 μg/ml). Cultures were fed every three days byreplacement of half the supernatant with fresh media. In general, wellswere of sufficient lymphoblastoid cell density between 2 and 3 weekspost-plating for analysis of anti-E. coli and S. marcescens serotypespecificity.

Thus, in this experiment one cloned transformed human cell line wasachieved which is continuous (immortal) and secretes a human monoclonalantibody to a determinant on the surface of the E. coli and S.marcescens serotypes set forth.

Prior to filing of this patent application, the continuous transformedhuman cell line identified as 7D7 was deposited with the American TypeCulture Collection, Rockville, Md. as A.T.C.C. No. CRL 9009.

E. Further Characterization of Intergenus Cross-Reactivity

Antibody from the cloned 7D7 cell line was assayed for furtherintergenus cross-reactivity by a modification of the standardimmunoblotting technique. Specifically, cross-reactivity to the bacteriaK. pneumoniae, E. aerogenes, and E. cloacae was investigated by spottingbacteria onto a gridded nitrocellulose paper disc, reacting the disccontaining the bacteria with said antibody, and developing the antibodyreactions with an alkaline pnosphatase/nitroblue tetrazolium enzymesystem. Briefly, 1.0 μl of bacteria (O.E.₆₆₀ =0.4) was spotted per gridsection of a nitro-cellulose paper disc (Schleicher and Schuell, 37 mmnitrocellulose disc, gridded, 0.45 μm). Each disc can conveniently hold60 different bacterial samples. The spotted discs were air-dried, fixedin 25% v/v isopropanol for 30 min, and blocked for 10 min in the non-fatdry milk reagent as described for the ELISA method. The blocked discswere washed three times for 5 min each in PBS/Tween 20 and weretransferred to the lids of 35×10 mm tissue culture dishes. The antibodycontaining supernatant (1.0 ml) was added to the lid and was incubatedat RT for 60 min. Following three 5 min washes in PBS/Tween 20, 1-2 mlof 1:1000 diluted (PBS) alkaline phosphatase conjugated goat-anti-humanimmunoglobulin (TAGO, Burlingame, Calif.) was added for 60 min at RT.The discs were washed as above, and were submerged in 1-2 ml of freshsubstrate prepared as follows: 16.5 mg of bromo-chlorindolylphosphateand 8.5 mg nitroblue tetrazolium were dissolved in 50 ml of alkalinephosphatase buffer (0.1M Tris-HCl, pH 9.5 with 0.1M NaCl and 5 mMMgCl₂), the solution was kept in the dark, and filtered immediatelybefore using. After appropriate color development (10-15 min), thereaction was quenched by rinsing the disc in several changes ofdistilled water. The developed discs can be stored after drying.

The discs contained; 50 K. pneumoniae capsule-typed reference strainsobtained from Dr. George Kenney, University of Washington, Department ofPathobiology, Seattle, Wash. and the American Type Culture Collection, 4E. aerogenes clinical blood isolates, and 6 E. cloacae clinical bloodisolates (blood isolates obtained from Harborview Hospital, Seattle,Wash.). The Enterobacter isolates were typed (speciated) using the API20E System of 23 standardized biochemical tests (API Analytab Products,Plainview, N.Y.). This method identities the genus and species ofgram-negative bacteria, but not the serotype. Therefore, theEnterobacter, unlike the E. coli, S. marcescens, and K. pneumoniae, arenot identified as to serotype, but only as to genus and specie.

From these experiments, the 7D7 antibody was observed to possess furtherintergenus cross-reactivity. This antibody reacted with the followingserotypes:

    ______________________________________                                        K. pneumoniae                                                                            E. coli   S. marcescens                                                                            E. aerogenes                                  ______________________________________                                        K14,57,60  08,75     012,13,15  Clinical Isolates                             ______________________________________                                    

Thus, the human monoclonal antibody 7D7 was observed to possessintergenus cross-reactivity to bacteria belonging to the species E.coli, S. marcescens, K. pneumoniae, E. aerogenes but not E. cloacae.

F. Characterization of Monoclonal Antibodies

The finding that the Monoclonal Antibody cross-reacted with severaldifferent bacterial genera suggested the antibody was directed against ashared protein or carbohydrate. These two molecular species have beenshown to account for intragenus cross-reactions (Mutharia, L. andHancock, R. E. W., "Characterization of Two Surface-Localized AntigenicSites on Porin Protein F of Pseudomonas aeruginosa," Canadian J. ofMicrobiol., 31:381-386, (1985) and Orskov, F. and Orskov, I.,"Serotyping of Escherichia Coli, " in Methods in Microbiology, Vol. 14,Bergan, T., ed. Academic Press, Orlando, Fla., 43-112 (1984)).

Biochemical characterization of the molecular species recognized by the7D7 antibody was accomplished by immunoblot analysis. Briefly, washedbacteria from a 20 ml overnight broth culture (for E. coli, S.marcescens, and E. aerogenes serotypes) or from overnight grown plates(K. pneumoniae) were extracted in 1.0 ml of a solution containing 64 mlof 50 mM Tris pH 7.6, 30 ml of glycerol, 0.3 gm of deoxycholate (sodiumsalt), 0.14 ml beta-mercaptoethanol, and 6 ml deionized water(Schechter, I. and Block, K., "Solubilization and Purification oftrans-Formesyl Pyrophosphate-Squalene Synthetase, "J. Biol Chem.,246:7690-7696, (1971)). After 18 hour incubation at 4° C., thesuspension was centrifuged at 10,000×g for 10 min. The supernatant wasremoved and the protein was quantitated using the Bio-Rad Protein Assay(Bio-Rad Laboratories, Richmond, Calif.). Between 100 and 1000 ng ofprotein (varies for each bacterial extract) from each bacteria were eachsubjected to sodium dodecyl sulphate (SDS)--polyacrylamide gelelectrophoresis (Kusecek, B., et al., "Lipopolysaccharide, Capsule, andfimbriae as Virulence Factors Among 01, 07, 016, 018 or 075 and K1, K5or K100 Escherichia coli," Infection and Immunity, 43:368-379 (1984)).Separated molecular species were transferred from the gel to anitrocellulose membrane (NCM) as described elsewhere (Towbin, H., etal., "Electrophoretic Transfer of Proteins From Polyacrylamide Gels toNitrocellulose Sheets: Procedure and Some Applications," Proc. Natl.Acad. Sci., 76:4350-4354, (1979)) and the NCM blot blocked for 1 hour inPBS-Tween (Batteiger, B, et al., "The Use of Tween 20 as a BlockingAgent in the Immunological Detection of Proteins transferred toNitrocellulose Membranes," J. Immunol. Meth. 55:297-307, (1982)). Theblot was incubated for 1 hour at RT in 10 ml of spent culturesupernatant from the 7D7 line. Following four 5 min rinses in PBS-Tween,the blot was incubated in goat anti-human Ig conjugated to alkalinephosphatase and developed as described herein for thebacteria-nitrocellulose disc assay. Positive reactions were noted in alltracks that contained deoxycholate extracts of the bacteria describedherein. In these tracks the 7D7 antibody appeared to recognize a seriesof regularly spaced molecular entities giving rise to a ladder-likepattern on the immunoblot. This profile was entirely consistent withthat seen in polyacrylamide gel electrophoretic analysis of LPS in thepresence of SDS, where it has been demonstrated that the heterogeneoussize profile exhibited by the bands is due to a population of LPSmolecules differing by weight increments equivalent to the number ofO-antigenic oligosaccharide side chain units present per molecule(Pavla, E. T. and Makela, P. H., "Lipopolysaccharide Heterogeneity inSalmonella typhimurium Analyzed by Sodium Dodecyl Sulfate/PolyacrylamideGel Electrophoresis," Eur. J. Biochem., 107:137-143, (1980) and Goldman,R. C. and Leive, L., "Heterogeneity of Antigenic-Side-Chain Length inLipopolysaccharide from Escherichia coli 0111 and Salmonella typhimuriumLT2, Eur. J. Biochem., 107:143-154, (1980)). These data indicate thatthe monoclonal antibody 707 is directed against an antigenic determinantshared by the LPS molecules found on some serotypes of E. coli, S.marcescens, K. pneumoniae, and E. aerogenes.

To further define the molecular nature of the antigen, the deoxycholateextracts were treated, prior to their electrophoresis, with theproteolytic enzyme Proteinase K (Eberling, W., et al., "Proteinase Kfrom Tritirachium album Limber," Eur. J. Biochem. 47:91-97 (1974)). Toprepare the sample, 10 μg of Proteinase K was added to 50 μg of sampleprotein and the mixture was heated to 65° C. for 60 min. The sampleswere electrophoresed and immunoblotted as described herein. Theimmunoblot patterns observed after Proteinase K treatment were identicalto those patterns observed without treatment and thus suggest that theantigen reactive with the 7D7 antibody is not protein in nature.

To specifically address whether 7D7 reacted with a carbohydrate epitope,the electrotransferred deoxycholate sample was subjected to mildperiodate oxidation prior to reacting the nitrocellulose paper withantibody. This reaction has been shown to destroy carbohydratedeterminants, and thus their subsequent reactivity with antibody,without altering protein or lipid epitopes (Woodward, M. P., et al.,"Detection of Monoclonal Antibodies Specific for Carbohydrate EpitopesUsing Periodate Oxidation," J. of Immunol. Methods, 78:143-153, (1985)).Briefly, after the electro-blotted nitrocellulose paper containing theelectrophoresed sample was blocked with PBS-Tween, as described herein,the paper was rinsed with 50 mM acetic acid sodium acetate pH 4.5buffer. The nitrocellulose paper was incubated in 50 mM periodic aciddissolved in the acetic acid buffer for 60 min in the dark at RT. Thetreated paper was rinsed three times in PBS-Tween and reacted with theantibody as described herein. Electro-blotted deoxycholate extractstreated in this manner were no longer reactive with the 7D7 monoclonalantibody. These data strongly indicate that the epitope recognized bythis antibody is a carbohydrate present on the LPS molecule of thebacteria described here.

The isotype of the 7D7 monoclonal antibody was determined in an ELISAprocedure similar to the specificity tests described above except thatbiotinylated goat anti-human IgG (gamma-chain specific, TAGO) orbiotinylated goat anti-human IgM (mu-chain specific, TAGO) was used asthe second step reagent instead of the more broadly reactivebiotinylated goat anti-human Ig. Both reagents were used at a 1:500dilution and the antigen plate contained pools of PLL immobilized E.coli 08 and 075 strains. Positive reaction of the 7D7 monoclonalantibody with the E. coli strains was observed only with the anti-IgMreagent, demonstrating an IgM isotype for the monoclonal antibody. Itwill be appreciated by those skilled in the art that if the aboveprocess were repeated several times and the isotypes of intergenuscross-reactive monoclonal antibodies so obtained were determined, onewould find additional e.g., IgM and IgG isotypes (Frosch, M., et al;,"NZB Mouse System For Production of Monoclonal Antibodies to WeakBacterial Antigens: Isolation of an IgG Antibody to the PolysaccharideCapsules of Escherichia coli K1 and Group B Meningocci," Proc. Natl.Acad. Sci., 82:1194-1198, (1985)).

G. In Vitro Activity

In vitro functional activity of the 7D7 monoclonal antibody was examinedin an in vitro opsonophagocytosis assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement.

Bacteria were prepared by either inoculating 10 ml of tryptic soy broth(TSB) with 50 μl of an overnight broth culture (for E. coli, S.marcescens, and E. aerogenes) or for K. pneumoniae, streaking a petridish containing Worfel-Ferguson Agar. For broth cultures, the tubes wereincubated at 37° C. on a shaker for 3 hours at which time 1.5 ml of theculture was centrifuged for 1 min at 10,000×g, the spent culture mediadiscarded, and the pellet was suspended in 3.5 ml of Hank's balancedsalt solution containing 0.1% gelatin and 5 mM HEPES (HBSS/Gel). For theagar plate grown bacteria, the colonies were scraped off the plate intosterile HBSS/Gel. The bacterial concentrations for bacteria grown underboth conditions were adjusted to 3×10³ bacteria/ml by measuring theO.D.₆₆₀ and making the appropriate dilutions (approximately 1:50,000).Human neutrophils were isolated according to van Furth and Van Zwet ("InVitro Determination of Phagocytosis and Introcellular Killing byPolymorphonuclear and Mononuclear Phagocytes," in Handbook ofExperimental Immunology, Vol. 2, D. M. Weir, ed., 2nd edition, BlackwellScientific Publications, Oxford, 36.1-36.24 (1973)) with severalmodifications. Buffy coat from 10 ml of heparinized blood was underlayedwith Ficoll-Pacque and centrifuged. The red blood cell (RBC) pellet waswashed once with RPMI 1640 medium and resuspended in an equal volume of37° C. PBS. Three ml of this suspension was added to 6 ml of 2% dextran(in 37° C. PBS) and the contents gently but thoroughly mixed end overend. After a 20 min incubation at 37° C. to allow the RBC's to sediment,the supernatant (containing neutrophils) was removed, washed twice in 4°C. PBS, once in HBSS/Gel, and suspended in same to 5×10⁷ neutropnils/ml.For the complement source used with E. coli and S. marcescens, humanserum was twice adsorbed with live bacteria pools (Bjornson, A. B. andMichael, J. G., "Factors in Human Serum Promoting Phagocytosis ofPseudomonas aeruginosa I. Interaction of Opsonins with the Bacterium,"J. of Inf. Dis., 130Suppl:S119-S126 (1974)) corresponding to theorganisms used in the assay. This serum was further adsorbed with boiledZymosan (Bjornson, supra) to remove the serum component properidin, amolecule necessary for the activation of the alternate complementpathway. For opsonophagocytic assays using K. pneumoniae and E.aerogenes, the complement source was unadsorbed normal human serum usedat 1% final concentration.

Plates used to quantify the number of surviving/destroyed bacteria wereprepared beginning with warming of 24 well plates at 37° C. for 3-5hours. A 0.4% solution of agarose in TSB was prepared by autoclaving themixture for 5 min and allowing it to cool to 50° C. in a water bath.Approximately 15 min before the end of the final incubation period inthe opsonophagocytosis assay, a 24 well plate was removed from the 37°C. incubator, placed on a 42° C. hot plate and 0.4 ml of TSB/agarose wasadded to each well. The plate was immediately returned to the 37° C.incubator such that the agarose never cooled below 37° C.

For the assay, 25 μl of 7D7 culture supernatant and 25 μl of anappropriate bacterial strain were added in duplicate to 96 well roundbottom microtiter plates and incubated at RT for 30 min. This wasfollowed by the addition of 15 μl of human complement, 15 μl of humanneutrophils (5×10⁶ /ml), and 70 μl HBSS/Gel. The entire surface of theplate was wiped with a sterile cotton swab, an adhesive plastic platesealer was applied to securely cover the entire plate and interwellareas, and the plate was rotated at 37° C. for 1 hour. After incubation,the plate was centrifuged for 5 min at 100×g, the plate sealer wasgently removed, and the plate surface was dried with a sterile cottonswab dipped in 70% ethanol. Fifty microliters was removed from eachmicrotiter well and was added to individual wells of the 24 wellquantitation plates which already contained the 0.4 ml/well of melted(38°-40° C.) 0.4% TSB/agarose. These plates were placed on a flatbedshaker for 1 min at 150 RPM and the agarose was allowed to harden for 15min at RT. Finally, a 0.4 ml TSB/agarose overlay was added to each well,followed by a hardening period of 15 min at 4° C. before the plates wereincubated overnight at 37° C. After 18 hours the colonies wereenumerated and the data was reported as colony forming units (CFU) foreach condition.

The bacterial serotypes used herein, except K. pneumoniae, were onlyinactivated in the presence of monoclonal antibody 7D7, an activecomplement source, and human neutrophils (Table 1). When this experimentwas repeated with several non-7D7 reactive bacterial serotypes, nobacteria destruction was observed (data not shown), thus demonstratingthe functional specificity of monoclonal antibody 7D7 and its capacityto opsonize bacteria and promote their phagocytosis. Since the combinedactions of opsonins (specific antibodies) and polymorphonuclearleukocytes (neutrophils) appeared to be the primary mechanism forimmunity to these bacterial strains, these data suggested that antibody7D7, would, after appropriate administration, provide protection againstlethal challenges with the bacteria serotypes described herein.

                  TABLE 1                                                         ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        E. coli  +        7D7       -.sup.a                                                                              0                                          08 and 075                                                                    E. coli  +        .sup. 6F11.sup.b                                                                       +       0                                          08 and 075                                                                    E. coli  -        7D7      +       0                                          08 and 075                                                                    E. coli  +        7D7      +       85%                                        08 and 075                                                                    S. marcescens                                                                          +        7D7      -       0                                          012 and 015                                                                   S. marcescens                                                                          +        6F11     +       0                                          012 and 015                                                                   S. marcescens                                                                          -        7D7      +       0                                          012 and 015                                                                   S. marcescens                                                                          +        7D7      +       85%                                        012 and 015                                                                   E. aerogenes                                                                           +        7D7      -       0                                          Isolates (2)                                                                  E. aerogenes                                                                           +        6F11     +       0                                          Isolates (2)                                                                  E. aerogenes                                                                           -        7D7      +       0                                          Isolates (2)                                                                  E. aerogenes                                                                           +        7D7      +       85%                                        Isolates (2)                                                                  ______________________________________                                         .sup.a (-) = heatinactivated (56° C. for 30 min) human complement.     .sup.b (6F11) = culture supernatant containing an IgM human monoclonal        antibody to Pseudomonas aeruginosa Fisher type 2.                        

H. In Vivo Activity

To test the above hypothesis, animal protection studies were performedwith the 7D7 antibody and at least one organism from the E. coli and E.aerogenes species described herein. 7D7 and negative control (6F11,human IgM monoclonal antibody specific to Pseudomonas aeruginosa Fisherimmunotype 2) antibodies were first concentrated from spent culturesupernatants by precipitation with solid ammonium sulphate (50% finalconcentration) (Good, A. J. et al., "Purification of Immunoglobulins andTheir Fragments," in Selected Methods in Cellular Immunology, Mishell,B. B. and Shiigi, S. M., eds., W. H. Freeman and Company, San Francisco,Calif. (1980) 279-286). Precipitated material was reconstituted insterile water and extensively dialyzed against PBS.

The IgM antibody in the ammonium sulphate salt precipitate was purifiedby affinity chromatography on a murine monoclonal anti-human IgMantibody affinity column. To prepare the column, one gram of dehydratedcyanogen bromide activated Sepharose 4B (Pharmacia) was mixed with 15 mlice cold 1 mM HCl in distilled water. The hydrated gel was washed in 30ml coupling buffer (0.1M carbonate (NaHCO₃) in 0.5M NaCl, pH 8.2),drained to form a moist cake and was combined with the ammonium saltprecipitate dissolved in 1-3 ml of coupling buffer. The gel suspensionwas mixed end-over-end for 2 hr at RT and subsequently centrifuged at200×g for 5 min. To block still available reactive sites, thesupernatant was removed, 10 ml of 1M ethanolamine was added to the gel,and mixing was continued as above. The suspension was centrifuged at200×g for 5 min and the supernatant was discarded. The gel was preparedfor use with 1 wash in 0.1M acetate/saline buffer (6.8 g sodium acetatetrihydrate and 14.6 g NaCl were dissolved in 500 ml distilled watercontaining 2.9 ml glacial acetic acid, pH 4.0), two washes in couplingbuffer, and two washes in PBS. The gel was poured into a PharmaciaC10/10 column and stored at 4° C. until use.

To purify the immunoglobulin, 0.5 ml of salt fractionated material wasdiluted to 2.0 ml in PBS and was added to the affinity column. Followingsample loading, the column was washed with PBS, pH 8.0 until theabsorbancy monitor indicated no further protein in the flow-through. Thebound antibody was eluted with 2 M MgCl₂ in PBS, the proteinconcentration was determined for each fraction at O.D.₂₈₀, and the peakfractions pooled. The antibody containing fraction was desalted on aG-25 Sephadex column and, if necessary, was con- centrated bymicroconcentration centrifugation (Centricon 30, Amicon Corp., Danver,Mass.) to 1-2 mg/ml. The final preparation was tested for purity bySDS-polyacrylamide gel electrophoresis followed by silver nitratestaining of proteins (Morrissey, J. H., "Silver Stain for Proteins inPolyacrylamide Gels: A Modified Procedure with Enhanced UniformSensitivity," Anal. Biochem., (1981) 117:307-310), and for antibodyactivity by ELISA as stated herein.

For each bacteria challenge, female, outbred Swiss-Webster mice weighingbetween 20 and 22 gm were divided into three groups of ten mice each. Arepresentative experiment was performed as follows:

    ______________________________________                                        Group        Bacteria     Antibody                                            ______________________________________                                        A            E. coli 08   7D7                                                 B            E. coli 08   6F11                                                C            S. marcescens 014                                                                          7D7                                                 ______________________________________                                    

Each group receiving antibody was injected intravenously with 200 μl ofsterile PBS containing 25 μg of purified antibody. Two hours later, allanimals were challenged intraperitoneally with 300 μl of live bacteriacontaining 3 LD₅₀ of their respective bacterial strain. The bacterialsuspension had been prepared from a broth culture in logarithmic phasegrowth, from which the bacteria was centrifuged, washed twice in PBS,and resuspended to the appropriate concentration in PBS. Animals wereobserved for a period of five days. Twenty-four to forty-eight hourspost-challenge all animals in Group B (irrelevant antibody) and Group C(irrelevant organism) were dead. In contrast, those animals that hadreceived the 7D7 (Group A) antibody were all alive and symptom free.

Animal protection models were used to test the protective effect of the7D7 antibody against bacterial challenges with organisms belonging tothe three genera stated in Table 1. A summary of the E. coli test datais presented in Table 2.

                  TABLE 2                                                         ______________________________________                                                                Survival/                                             Challenge Bacteria                                                                         Antibody   Challenge                                                                              % Survival                                   ______________________________________                                        E. coli 08   7D7        10/10    100                                          E. coli 08   6F11.sup.a 0/10     0                                            S. marcescens 014.sup.b                                                                    7D7        0/10     0                                            ______________________________________                                         .sup.a 6F11 antibody is specific to Pseudomonas aeruginosa Fisher             immunotype 2 and serves as negative control antibody.                         .sup.b S. Marcescens 014 is not reactive with the 7D7 antibody and serves     as a nonspecific control organism.                                       

                  TABLE 2a                                                        ______________________________________                                               % Survival.sup.c                                                              Day                                                                    Antibody 0          1     2        3   4                                      ______________________________________                                        6F11     12          2    2        1   1                                      7D7      12         10    6        4   4                                      ______________________________________                                         .sup.c LD.sub.50 's and protection studies were performed with mice           rendered neutropenic by injection with cyclophosphamide as follows: Day 0     150 mg/kg, Day 2  50 mg/kg. On Day 4 mice received antibody and bacteria      as described herein.                                                     

The data and data obtained with Enterobactor aerogenes demonstrate thatthe human monoclonal antibody 7D7 is able to protect mice from lethalchallenges with bacteria belonging to two different gram-negativebacteria species. Because 7D7 is reactive with a carbohydrate epitopepresent on LPS, but LPS molecules on K. pneumoniae are less accessible(Orskov, I. and Orskov, F , "Serotyping of Klebsiella," in Methods inMicrobiology, Vol. 14, Bergan, T., Ed., Academic Press, Orlando, Fla.(1984) 143-146) protection by the 7D7 antibody against K. pneumoniaeinfections was not evident (data not shown). Nor was the 7D7 antibodyprotective against a strain of Serratia marcescens tested (data notshown). Nonetheless, the intergenus cross-reactive human monoclonalantibody 7D7 was able to afford protection with 25 μg of purifiedantibody against infection by organisms belonging to the gram-negativebacteria species E. coli and E. aerogenes.

EXAMPLE II

Example II demonstrates methods for the production and selection of ahuman monoclonal antibody that possesses intergenus cross-reactivityagainst members of the species Serratia marcescens, Klebsiellapneumoniae and Enterobacter aerogenes. Further, this exampledemonstrates the in vitro opsonic activity of said antibody againsthomologous S. marcescens, K. pneumoniae, and E. aerogenes serotypes. Theprocess of Example I (essentially as described in parts A through G) wasrepeated to produce a human monoclonal antibody that wascross-protective against infections caused by the bacteria describedherein, except that it was necessary to make specific modifications tocharacterize and assay the antibody described in this Example. Thefollowing are changes in assay procedures and the results obtained withthe monoclonal antibody described herein.

1. Culture supernatants from six transformations were analyzed by theabove method resulting in the identification of one well (4F10) whichpossessed activity on the S. marcescens serotype plate, but not on theE. coli or control plate. It was determined in subsequent ELISA's withindividual S. marcescens serotypes, that this well contained antibodyreactive with the S. marcescens serotypes 015 and 018, but not any otherof the twenty known S. marcescens LPS serotypes.

Prior to the filing of this patent application, the continuoustransformed human cell line identified as 4F10 was deposited with theAmerican Type Culture Collection, Rockville, Md. as A.T.C.C. No. CRL9007.

2. Antibody from the cloned 4F10 cell line was assayed for furtherintergenus cross-reactivity by a modification of the standardimmunoblotting technique. Specifically, cross-reactivity to the bacteriaK. pneumoniae, E. aerogenes, and E. cloacae was investigated by spottingbacteria onto a gridded nitrocellulose paper disc, reacting the disccontaining the bacteria with said antibody, and developing the antibodyreactions with an alkaline phosphatase/nitroblue tetrazolium enzymesystem.

From these experiments, the 4F10 antibody was observed to possessfurther intergenus cross-reactivity. This antibody reacted with thefollowing serotypes:

    ______________________________________                                        K. pneumoniae  S. marcescens                                                                            E. aerogenes                                        ______________________________________                                        K3,12,29,31,68,72                                                                            015,18     Clinical Isolates                                   ______________________________________                                    

Thus, the human monoclonal antibody 4F10 possessed intergenuscross-reactivity to bacteria belonging to the species S. marcescens, K.pneumoniae, E. aerogenes.

3. Using the immunoblot technique, positive reactions were noted in alltracks that contained deoxycholate extracts of the bacteria describedherein. In these tracks, the 4F10 antibody appeared to recognize eithera broad band of components or a series of regularly spaced molecularentities giving rise to a ladder-like pattern. This profile was entirelyconsistent with that seen in polyacrylamide gel electrophoretic analysesof carbohydrate moieties that demonstrate either extensive molecularweight heterogeneity due to an often repeating specific sugar sequenceor to LPS molecules differing by weight increments equivalent to thenumber of O-antigenic oligosaccharide side chain units per molecule(Vimr, E. R., et al., "Use of Procaryotic-Derived Probes to IdentifyPoly (Sialic Acid) in Neonatal Neuronal Membranes," Proc. Natl. Acad.Sci., (1983) 81:1971-1975; and Holden, K. G. et al., "GelElectrophoresis of Mucous Glycoproteins, I. Effect of Gel Porosity,"Biochemistry (1971) 10:3105-3109). These data indicate that themonoclonal antibody 4F10 is directed against an antigenic determinantshared by carbohydrate molecules found on some serotypes of S.marcescens, K. pneumoniae, and E. aerogenes.

The immunoblot patterns observed after Proteinase K treatment wereidentical to those patterns observed without treatment and thus suggestthat the antigen reactive with the 4F10 antibody is not protein innature.

To specifically address whether 4F10 reacted with carbohydrate epitope,the electrotransferred deoxycholate sample was subjected to mildperiodate oxidation prior to reacting the nitrocellulose paper withantibody. Electro-blotted deoxycholate extracts treated in this mannerwere no longer reactive with the 4F10 monoclonal antibody. These datastrongly indicate that the epitope recognized by this antibody is acarbohydrate moiety present on molecules possessed by the bacteriadescribed herein.

4. The isotype of the 4F10 monoclonal antibody was determined in anELISA procedure similar to the specificity tests described in Example Iexcept that the antigen plate contained a pool of PLL immobilized S.marcescens 015 and 018 serotypes. Positive reaction of the 4F10monoclonal antibody with the S. marcescens serotypes was observed onlywith the anti-IgM reagent, demonstrating an IgM isotype for themonoclonal antibody. It will be appreciated by those skilled in the artthat if the process of this example were repeated several times and theisotypes of intergenus cross-reactive monoclonal antibodies so obtainedwere determined, one would find additional isotypes, e.g., IgM and IgGisotypes.

5. In vitro functional activity of the 4F10 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement.

The bacterial serotypes used herein were only inactivated in thepresence of monoclonal antibody 4F10, an active complement source, andhuman neutrophils (Table 3). When this experiment was repeated withseveral non-4F10 reactive bacterial serotypes, no bacterial destructionwas observed (data not shown) thus demonstrating the functionalspecificity of monoclonal antibody 4F10 and its capacity to opsonizebacteria and promote their phagocytosis.

                  TABLE 3                                                         ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        S. marcescens                                                                          +        4F10      -.sup.a                                                                              0                                          018 and 015                                                                   S. marcescens                                                                          +        .sup. 6F11.sup.b                                                                       +       0                                          018 and 015                                                                   S. marcescens                                                                          -        4F10     +       0                                          018 and 015                                                                   S. marcescens                                                                          +        4F10     +       85%                                        018 and 015                                                                   K. pneumoniae                                                                          +        4F10     -       0                                          K3 and K12                                                                    K. pneumoniae                                                                          +        6F11     +       0                                          K3 and K12                                                                    K. pneumoniae                                                                          -        4F10     +       0                                          K3 and K12                                                                    K. pneumoniae                                                                          +        4F10     +       60%                                        K3 and K12                                                                    E. aerogenes                                                                           +        4F10     -       0                                          Isolates (2)                                                                  E. aerogenes                                                                           +        6F11     +       0                                          Isolates (2)                                                                  E. aerogenes                                                                           -        4F10     +       0                                          Isolates (2)                                                                  E. aerogenes                                                                           +        4F10     +       85%                                        Isolates (2)                                                                  ______________________________________                                         .sup.a (-) = heatinactivated (56° C. for 30 min) human complement.     .sup.b (6F11) = culture supernatant containing an IgM human monoclonal        antibody to Pseudomonas aeruginosa Fisher type 2.                        

EXAMPLE III

Example III demonstrates methods for the production of a humanmonoclonal antibody that is reactive with both the Escherichia colicapsular type K1 and Neisseria meningitidis (N. meningitidis) Group Bpolysaccharide and further demonstrates the protective activity of saidantibody in vivo against a lethal challenge of homologous E. coli and N.meningitidis bacterial species. The process of Example I (essentiallydescribed in parts A through G) was repeated to produce a humanmonoclonal antibody that was cross-protective against infections causedby the bacteria described herein, except that it was necessary to makespecific modifications to characterize and assay the antibody describedin this Example. The following are changes in assay procedures and theresults obtained with the monoclonal antibody described herein.

1. Culture supernatants from five transformations were analyzed by theabove method resulting in the identification of four wells (5D4, 2C10,9B10, and 8A8) which contained anti- E. coli specificity on the E. coliserotype plate, but not on the S. marcescens or control plates. It wasdetermined in subsequent ELISA's, conducted as set forth, on individualE. coli serotypes, that these wells contained antibody reactive with, atleast, the E. coli serotypes: 01 (ATCC 23499) , 07:K1 (ATCC 12792) ,016:K1 (ATCC 23511) , and 050 (CDC 1113-83), but not 04 (ATCC 12792),06:K2 (ATCC 19138), 08:K8 (ATCC 23501), 09:K9 (ATCC 23505), or 022:K13(ATCC 23517). Due to its better performance during the cloning procedureand increased antibody production, the 9B10 monoclonal antibody wasselected for further analysis.

Prior to filing of this patent application the continuous transformedhuman cell line identified herein as 9B10 was deposited in the AmericanType Culture Collection, Rockville, Md. as A.T.C.C. No. CRL 9006.

2. The finding that the monoclonal antibodies from each of the clonesreacted with the identical group of E. coli O-antigen serotypes,indicated that these antibodies were directed against a bacterialsurface structure common to these serotypes. Several approaches wereused to define the surface structure common to these E. coli serotypes.As set forth, two (07:K1 and 016:K1) of the four E. coli serotypesidentified by the 9B10 antibody possessed the K1 capsular serotype,while the other two (01 and 050) had not been typed for their K-antigenserotype. Thus, the possibility was pursued that the 9B10 antibodycontained reactivity to the K1 antigen and that the E. coli strainspossessing the O-antigen serotypes 01 and 050 also possessed the K1capsular serotype.

Others have taken advantage of the thermolability of the K1 capsule toestablish its presence. Heating of K1 positive E. coli serotypes in aboiling water bath at 100° C. for 60 minutes removes the subsequentability of these strains to react with anti-K1 sera and enhances theirability to react with anti-O antigen sera (Orskov, F. and Orskov, I.,"Serotyping of Escherichia coli," in Methods in Microbiology, Vol. 14,T. Bergan, ed., Academic Press, London (1984) pp. 44-105). Thereciprocal effects to boiling are most likely due to the removal of thecapsule and the increased accessibility of antibody forlipopolysaccharide (LPS) molecules. The E. coli K1 positive serotypes(07 and 016) and the non-K1 typed serotypes (01 and 050) were heated asset forth and reacted with the 9B10 antibody and LPS serotype specificheterologous sera (Difco Bacto-E. coli Typing Reagents) in the ELISAprocedure. Heat treated organisms lost all reactivity to the 9B10antibody and had increased their reactivity with their homologous LPSserotype specific sera, while non-treated (control) organisms remainedstrongly reactive with 9B10 culture supernatants and poorly reactivewith their respective LPS serotype specific antisera.

The polysaccharide (carbohydrate) from Neisseria meningitidis Group Bbacteria (a homopolymer of sialic acid, alpha 2,8-linked poly-N-acetylneuraminic acid) has been proven to be chemically and antigenicallyhomogeneous with the E. coli K1 polysaccharide (Grados, O. and Ewing, W.H., "Antigenic Relationship between Escherichia coli and Neisseriameningitidis, "J. Immunol. (1973) 110:262-268). These data suggest thatif the 9B10 monoclonal antibody contains specificity to E. coli K1capsule then the antibody should also contain specificity to N.meningitidis Group B polysaccharide and further, that monoclonalantibodies containing specificity to the Group B polysaccharide of N.meningitidis should also demonstrate reactivity to E. coli strainspossessing the K1 capsule. Two experimental protocols were used thattested for (1) the ability of the 9B10 antibody to react with N.meningitidis and (2) the ability of an antibody against N. meningitidisGroup B polysaccharide to react with the four E. coli 9B10 reactiveserotypes.

Highly purified Group B polysaccharide (Connaught Laboratories, Toronto,Canada) and viable N. meningitidis Group B bacteria were reacted withthe 9B10 antibody in an ELISA as set forth. The 9B10 monoclonal antibodystrongly reacted against both antigen preparations. To prove theconverse specificity, a commercially available N. meningitidis Group BMeningitis Test Kit ("Directagen" Direct Antigen Detection System,Hynson, Westcott, and Dunning, Baltimore, Md.), that utilizes latexspheres coated with a murine monoclonal antibody to the Group Bpolysaccharide, was used. In agglutination assays using the 9B10positive E. coli serotypes, all four serotypes demonstrated strongreactivity with the antibody coated spheres. E. coli serotypes known tobe K1-antigen negative were also negative in this test system.Collectively, these data indicate that the 9B10 monoclonal antibody isreactive with the E. coli K1 capsule and the type-specific carbohydrateon N. meningitidis Group B. Further, since many of these assays wereperformed with intact, viable bacteria, it can be inferred thatmonoclonal antibody 9B10 is specific for some portion of an externallyexposed region of the poly-sialic acid molecule.

3. The isotype of the 9B10 monoclonal antibody was determined in anELISA procedure similar to the specificity tests described in Example Iexcept that the antigen plate contained a pool of PLL immobilized E.coli K1 positive serotypes. Positive reaction of the 9B10 monoclonalantibody with the K1 positive E. coli serotypes was observed only withthe anti-IgM reagent, demonstrating an IgM isotype for the monoclonalantibody. It will be appreciated by those skilled in the art that if theprocess of this Example were repeated several times and the isotypes ofK1-specific monoclonal antibodies so obtained were determined, one wouldfind additional isotypes, e.g., IgM and IgG isotypes.

4. In vitro functional activity of the 9B10 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement.

K1 positive E. coli serotypes were only inactivated in the presence ofmonoclonal antibody 9B10, an active complement source, and humanneutrophils (Table 4). When this experiment was repeated with several K1negative E. coli serotypes, no bacterial destruction assay was observed(data not shown) thus demonstrating the K1 specificity of monoclonalantibody 9B10 and its capacity to opsonize bacteria and promote theirphagocytosis. Since the combined action of opsonins (specificantibodies) and polymorphonuclear leukocytes (neutrophils) appeared tobe the primary mechanism for immunity to K1 positive E. coli serotypes,these data suggested that antibody 9B10, would, after appropriateadministration, provide protection against a lethal challenge with anyE. coli K1 encapsulated serotypes, regardless of its O-antigen serotype.

                  TABLE 4                                                         ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        E. coli 01:K1                                                                          +        9B10      -.sup.a                                                                              0                                          and 018:K1                                                                    E. coli 01:K1                                                                          +        .sup. 6F11.sup.b                                                                       +       0                                          and 018:K1                                                                    E. coli 01:K1                                                                          -        9B10     +       0                                          and 018:K1                                                                    E. coli 01:K1                                                                          +        9B10     +       99%                                        and 018:K1                                                                    N. meningitidis                                                                        +        9B10     -       0                                          Group B                                                                       N. meningitidis                                                                        +        6F11     +       0                                          Group B                                                                       N. meningitidis                                                                        -        9B10     +       0                                          Group B                                                                       N. meningitidis                                                                        +        9B10     +       99%                                        Group B                                                                       ______________________________________                                         .sup.a (-) = heatinactivated (56° C.) for 30 min) human complement     .sup.b (6F11) = culture supernatant containing an IgM human monoclonal        antibody to Pseudomonas aeruginosa Fisher type 2.                        

5. To test the above hypothesis, animal protection studies wereperformed with the 9B10 antibody and several K1 positive and K1 negativeE. coli serotypes, as well as a N. meningitidis Group B serotype (strainH313, obtained from Dr. Carl Frasch, Laboratory of BacterialPolysaccharides, Office of Biologics, Food and Drug Administration,Bethesda, Md.).

For each bacteria challenge, female, outbred Swiss-Webster mice weighingbetween 20 and 22 gm were divided into three groups of ten mice each. Arepresentative experiment was performed as follows:

    ______________________________________                                        Group          Bacteria Antibody                                              ______________________________________                                        A              E. coli K1                                                                             9B10                                                  B              E. coli K1                                                                             6F11                                                  C              E. coli K2                                                                             9B10                                                  ______________________________________                                    

Each group receiving antibody was injected intravenously with 200 μl ofsterile PBS containing 25 μg of purified antibody. Two hours later, allanimals were challenged intraperitoneally with 300 μl of live bacteriacontaining 3 LD₅₀ of their respective bacterial strain. The bacterialsuspension had been prepared from a broth culture in logarithmic phasegrowth, from which the bacteria was centrifuged, washed twice in PBS,and resuspended to the appropriate concentration in PBS. Animals wereobserved for a period of five days. Twenty-four to forty-eight hourspost-challenge all animals in Group B (irrelevant antibody) and Group C(irrelevant organism) were dead. In contrast, those animals that hadreceived the 9B10 (Group A) antibody were all alive and symptom free.

This animal protection model was used to demonstrate the therapeuticeffect of the 9B10 antibody against bacterial challenges with organismsbelonging to the two species stated herein. A summary of these data ispresented in TabLe 5.

                  TABLE 5                                                         ______________________________________                                                                Survival/                                             Challenge Bacteria                                                                          Antibody  Challenge % Survival                                  ______________________________________                                        E. coli K1    9B10      10/10     100%                                        E. coli K1     6F11.sup.a                                                                              0/10     0%                                          E. coli K2.sup.b                                                                            9B10       0/10     0%                                          N. meningitidis Group B                                                                     9B10      5/5       100%                                        N. meningitidis Group B                                                                     6F11      0/5       0%                                          E. coli K2    9B10      0/5       0%                                          ______________________________________                                         .sup.a 6F11 antibody is specific to Pseudomonas aeruginosa Fisher             immunotype 2 and serves as negative control antibody.                         .sup.b E. coli K2 is not reactive with the 9B10 antibody and serves as        nonspecific control organisms.                                           

These data demonstrate that the human monoclonal antibody 9B10 is ableto protect mice from lethal challenges with bacteria belonging to twodifferent gram-negative bacterial species. The intergenuscross-protective antibody was able to passively protect againstinfection by organisms belonging to the gram-negative bacterial speciesE. coli and N. meningitidis Group B.

EXAMPLE IV

Example IV demonstrates methods for the production of a human monoclonalantibody that possesses intergenus cross-reactivity against members ofthe species Escherichia coli (E. coli), Enterobacter cloacae (E.cloacae) and Group B Stretococcus. Further this Example demonstrates anantibody cross-reactive with species belonging to the two main bacteriadivisions; gram-negative (E. coli and E. cloacae) and gram-positive(Group B Streptococcus). Even further, this Example demonstrates the invivo protective activity of said antibody against a lethal challenge ofhomologous E. coli, and Group B Streptococcus serotypes. The process ofExample I (essentially described in parts A through G) was repeated toproduce a human monoclonal antibody that was cross-protective againstinfections caused by the bacteria described herein, except that it wasnecessary to make specific modifications to characterize and assay theantibody described in this Example. The following are changes in assayprocedures and the results obtained with the monoclonal antibodydescribed herein.

1. Supernatants were screened for the presence of anti-Group BStreptococcus antibodies using an enzyme-linked immunosorbent assay(ELISA) technique as described in Example I. The antigen platesconsisted of a series of flat-bottom 96-well Immunolon 2 microtiterplates, the wells of which contained mixtures of Group B Streptococcicapsule types affixed to the well surfaces with poly-L-lysine (PLL).Various antigen plates used in the screen included: (1) a mixture ofGroup B Streptococcus types Ia (A.T.C.C. No. 12400), Ib (A.T.C.C. No.12401), Ic (A.T.C.C. No. 27591); (2) a mixture of types II (A.T.C.C. No.12973) and III (clinical isolate obtained from Dr. C. Wilson, Children'sOrthopedic Hospital, Dept. Infectious Disease, Seattle, Wash.); and (3)a microtiter plate with no bacteria.

Culture supernatants from two transformations were analyzed by the abovemethod resulting in the identification of one well (4B9) which possessedactivity on both Group B Streptococcus typing plates, but not thecontrol plates. It was determined in subsequent ELISA's with individualGroup B Streptococcus types, that this well contained antibody reactivewith all five reference typing strains.

Thus, in this experiment one cloned transformed human cell line wasachieved which is continuous (immortal) and secretes a human monoclonalantibody to a determinant on the surface of the Group B Streptococcustypes set forth.

Prior to filing of this patent application, the continuous transformedhuman cell line identified as 4B9 was deposited with the American TypeCulture Collection, Rockville, Md. as A.T.C.C. No. CRL 9008.

2. Antibody from the cloned 4B9 cell line was assayed forcross-reactivity to gram-negative and gram-positive bacteria by amodification of the standard immunoblotting technique. Specifically,cross-reactivity to the bacteria E. coli, K. pneumoniae, S. marcescens,E. aerogenes, E. cloacae, Hemophilus influenzae, and Staphylococcusaureus was investigated by spotting bacteria onto a griddednitrocellulose paper disc, reacting the disc containing the bacteriawith said antibody, and developing the antibody reactions with analkaline phosphatase/nitroblue tetrazolium enzyme system (as describedin Example I).

From these experiments, the 4B9 antibody was observed to possesscross-reactivity with particular gram-negative bacterial species. Thisantibody reacted with the E. coli LPS serotypes 04, 07, 018, and 025,and the E. cloacae clinical isolates. Thus the human monoclonal antibody4B9 possesses intergenus cross-reactivity between the gram-negative andgram-positive bacteria belonging to the species E. coli, E. cloacae, andGroup B Streptococcus.

3. The finding that the monoclonal antibody cross-reacted with severaldifferent bacterial genera belonging to both gram-positive andgram-negative bacterial divisions, suggested the antibody was directedagainst a shared protein or carbohydrate. The biochemicalcharacterization of the molecular species recognized by the 4B9 antibodywas accomplished by immunoblot analysis. For analysis of thegram-negative genera, washed bacteria were extracted in deoxycholate asdescribed in EXAMPLE I. For the gram-positive bacteria, 1.0 L ofbacteria cultured for 6 hours in modified Todd-Hewitt Broth (Difco,Todd-Hewitt Broth containing 2.8 gm/L anhydrous sodium phosphate, pH7.8) at 37° C. were harvested by centrifugation and washed three timesin PBS. The bacteria were resuspended in 85 ml of protoplast medium (40%sucrose w/v in 0.03M potassium phosphate buffer, pH 6.8 containing 10 mMMgCl₂) and the suspension was warmed to 37° C. for 10 min. Approximately3000 units of the mutanolysin (SIGMA) were added and the mixture wasshaken at 37° C. for 90 min or until the OD₆₆₀ of the suspension hadbeen reduced by >90%. The digested material was centrifuged at 2000×gfor 15 min at RT and the supernatant was dialyzed against PBS for 48 hr(Young, M. K. and Mattingly, S. J., "Biosynthesis of Cell WallPeptidoglycan and Polysaccharide Antigens by Protoplasts of Type IIIGroup B Streptococcus," J. Bact., (1983) 154:211-220). The dialysate wasconcentrated ten-fold by positive pressure dialysis through a PM-10filter (Amicon Corp., Danvers, Mass.).

Carbohydrates binding to wheat germ agglutinin were purified by affinitychromatography on a wheat germ lectin Sepharose 6MB column (SIGMA). Thebound digest, described herein, was eluted from the column with 10 ml of0.1M N-acetylglucosamine and the eluate was dialyzed against distilledwater at 4° C. The affinity purified eluate was dried by lyophilizationand the dry weight of the resulting material was obtained (Gray, B. M.,et al., "Interaction of Group B Streptococcal Type-SpecificPolysaccharides with Wheat Germ Agglutinin and Other Lectins," J. orImmunol. Meth., (1984) 72:269-277). Positive reactions were noted in alltracks that contained deoxycholate extracts of the bacteria describedherein. In those tracks containing extracts from gram-negative bacteria,the 4B9 antibody appeared to recognize a series of regularly spacedmolecular entities giving rise to a ladder-like pattern on theimmunoblot. This profile was entirely consistent with that seen inpolyacrylamide gel electrophoretic analysis of LPS in the presence ofSDS, where it has been demonstrated that the heterogenous size profileexhibited by the bands is due to a population of LPS molecules differingby weight increments equivalent to the number of O-antigenicoligosaccharide side chain units present per molecule (Pavla, E. T. andMakela, P. H., supra and Goldman, R. D. and Leive, L., supra). In thosetracks containing extracts from the Group B Streptococcus types, the 4B9antibody appeared to recognize components present on a broad band. Thisprofile was consistent with that seen in polyacrylamide gelelectrophoresis analyses of carbohydrate moieties that demonstrateextensive molecular weight heterogeneity with a frequently repeatingspecific sugar sequence (Vmir, E. R. et al., supra and Holden, K. G.,supra). These data indicate that the monoclonal antibody 4B9 is directedagainst an antigenic determinant shared by molecules found on someserotypes of E. coli, E. cloacae, and Group B Streptococcus.

To further define the molecular nature of the antigen, the deoxycholateextracts were treated with proteolytic enzyme Proteinase K prior totheir electrophoresis (Eberling, W., supra). The immunoblot patternsobserved after Proteinase K treatment were identical to those patternsobserved without treatment and thus suggest that the antigen reactivewith the 4B9 antibody is not protein in nature.

To specifically address whether 4B9 reacted with a carbohydrate epitope,the electrotransferred deoxycholate and wheat germ agglutinationaffinity purified samples were subjected to mild periodate oxidationprior to reacting the nitrocellulose paper with antibody (see EXAMPLEI). Electro-blotted deoxycholate extracts treated in this manner were nolonger reactive with the 4B9 monoclonal antibody. These data stronglyindicate that that epitope recognized by this antibody is a carbohydratemoiety present in molecules possessed by both the gram-negative andgram-positive bacteria described herein.

4. The isotype of the 4B9 monoclonal antibody was determined in an ELISAprocedure similar to the specificity tests described above except thatthe antigen plate contained a pool of PLL immobilized Group BStreptococcus types II and III. Positive reaction of the 4B9 monoclonalantibody with the Group B streptococcus strains was observed only withthe anti-IgM reagent, demonstrating an IgM isotype for the monoclonalantibody.

5. In vitro functional activity of the 4B9 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement.

The bacterial strains used herein were only inactivated in the presenceof monoclonal antibody 4B9, an active complement source, and humanneutrophils (Table 6). When this experiment was repeated with severalnon-4B9 reactive bacterial serotypes, no bacteria destruction wasobserved (data not shown) thus demonstrating the functional specificityof monoclonal antibody 4B9 and its capacity to opsonize bacteria andpromote their phagocytosis. Since the combined actions of opsonins(specific antibodies) and polymorphonuclear leukocytes (neutrophils)appeared to be the primary mechanism for immunity to these bacterialstrains, these data suggest that antibody 4B9, would, after appropriateadministration, provide protection against lethal challenges with thebacteria strains described herein.

                  TABLE 6                                                         ______________________________________                                                  Neutro-                  % Destruction of                           Bacteria  phils   Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        E. coli   +       4B9       -.sup.a                                                                              0                                          018 and 025                                                                   E. coli   +       .sup. 6F11.sup.b                                                                       +       0                                          018 and 025                                                                   E. coli   -       4B9      +       0                                          018 and 025                                                                   E. coli   +       4B9      +       85%                                        018 and 025                                                                   E. cloacae                                                                              +       4B9      -       0                                          Isolates                                                                      E. cloacae                                                                              +       6F11     +       0                                          Isolates                                                                      E. cloacae                                                                              -       4B9      +       0                                          Isolates                                                                      E. cloacae                                                                              +       4B9      +       85%                                        Isolates                                                                      Group B. Strep.                                                                         +       4B9      -       0                                          Types Ia and III                                                              Group B. Strep.                                                                         +       6F11     +       0                                          Types Ia and III                                                              Group B. Strep.                                                                         -       4B9      +       0                                          Types Ia and III                                                              Group B. Strep.                                                                         +       4B9      +       85%                                        Types Ia and III                                                              ______________________________________                                         .sup.a (-) = heatinactivated (56° C. for 30 min) human complement.     .sup.b (6F11) = culture supernatant containing an IgM human monoclonal        antibody to Pseudomonas aeruginosa Fisher type 2.                        

6. To test the above hypothesis, animal protection studies wereperformed with the 4B9 antibody and at Least one organism from eachgenus described herein.

From each gram-negative bacteria challenge, female, outbredSwiss-Webster mice weighing between 20 and 22 gm were divided into threegroups of ten mice each. A representative experiment was performed asfollows:

    ______________________________________                                        Group        Bacteria     Antibody                                            ______________________________________                                        A            E. coli 018  4B9                                                 B            E. coli 018  6F11                                                C            S. marcescens 014                                                                          4B9                                                 ______________________________________                                    

Each group receiving antibody was injected intravenously with 200 μl ofsterile PBS containing 25 μg of purified antibody. Two hours later, allanimals were challenged intraperitoneally with 300 μl of live bacteriacontaining 3 LD₅₀ of their respective bacterial strain. The bacterialsuspension had been prepared from a broth culture in logarithmic phasegrowth, from which the bacteria was centrifuged, washed twice in PBS,and resuspended to the appropriate concentration in PBS. Animals wereobserved for a period of five days. Twenty-four to forty-eight hourspost-challenge all animals Group B (irrelevant antibody) and Group C(irrelevant organism) were dead. In contrast, those animals that hadreceived the 4B9 (Group A) antibody were all alive and symptom free.

For the Group B Streptococcus protection studies, a neonatal rat modelwas used. Outbred Sprague-Dawley rat pups (housed with their mothers),less than 48 hours old received antibody and bacteria essentially asdescribed for the mouse protection studies. Primary differences were asfollows: 1) both the antibodies and bacterial challenges were injectedintraperitoneally, and 2) the inoculum size was reduced to 20 μl.

These animal protection models were used to demonstrate the therapeuticeffect of the 4B9 antibody against bacterial challenges with organismsbelonging to two of the three species stated herein. A summary of thesedata is presented in Table 7.

                  TABLE 7                                                         ______________________________________                                                                Survival/                                             Challenge Bacteria                                                                          Antibody  Challenge % Survival                                  ______________________________________                                        E. coli 025   4B9       10/10     100                                         E. coli 025    6F11.sup.a                                                                             0/10      0                                           S. marcescens 014.sup.b                                                                     4B9       0/10      0                                           Group B       4B9       10/10     100                                         Streptococcus Ia and III                                                      Group B       6F11      0/10      0                                           Streptococcus Ia and III                                                      S. marcescens 014                                                                           9B10      0/10      0                                           ______________________________________                                         .sup.a 6F11 antibody is specific to Pseudomonas aeruginosa Fisher             immunotype 2 and serves as negative control antibody.                         .sup.b S. marcescens 014 is not reactive with the 4B9 antibody and serve      as a nonspecific control organisms.                                      

These data demonstrate that the human monoclonal antibody 4B9 is able toprotect mice and rats from lethal challenges with bacteria generabelonging to both gram-negative and gram-positive bacterial divisions.The intergenus cross-reactive human monoclonal antibody 4B9 was able toafford protection with 25 μg of purified antibody against infection byorganisms belonging to the gram-negative bacteria genera E. coli and thegram-positive bacteria belonging to Group B Streptococcus.

EXAMPLE V

EXAMPLE V demonstrates methods for the production and selection of ahuman monoclonal antibody that possesses intergenus cross-reactivityagainst members of the genera Serratia marcescens, Klebsiellapneumoniae, and Enterobacter aerogenes. Further, this exampledemonstrates the in vitro opsonic activity of said antibody againsthomologous S. marcescens, K. pneumoniae, and E. aerogenes serotypes. Theprocess of Example I (essentially as described in parts A through G) wasrepeated, except that it was necessary to make specific modifications tocharacterize and assay the antibody described in this Example. Thefollowing are changes in assay procedures and the results obtained withthe monoclonal antibody described herein.

1. Culture supernatants from four transformations were analyzed by theabove method resulting in the identification of one well (7E10) whichpossessed binding activity on at least one of four K. pneumoniaeserotype plates, containing the capsule serotypes; 1, 2, 3, 4, 6, 8, 9,19, 20, 21, 24, 27, 31, 43, 44, and 55, but not on the control plate (nobacteria). Antibody from the 7E10 cell line was assayed for furtherintergenus cross-reactivity by a modification of the standardimmunoblotting technique. Specifically, cross-reactivity to the bacteriaK. pneumoniae, E. aerogenes, S. marcescens, E. coli, and P. aeruginosawas investigated by spotting bacteria onto a gridded nitrocellulosepaper disc, reacting the disc containing the bacteria with saidantibody, and developing the antibody reactions with an alkalinephosphatase/nitroblue tetrazolium enzyme system.

From these experiments, the 7E10 antibody was observed to possessfurther intergenus cross-reactivity. This antibody reacted with thefollowing species and serotypes:

    ______________________________________                                        K. pneumoniae  S. marcescens                                                                            E. aerogenes                                        ______________________________________                                        K2,8,11,12,13,21,                                                                            04,12      Clinical Isolates                                   26,29,30,33,42,68,69                                                          ______________________________________                                    

Thus, the human monoclonal antibody 7E10 possessed intergenuscross-reactivity to bacteria belonging to the genera K. pneumoniae, S.marcescens, and E. aerogenes.

2. The isotype of the 7E10 monoclonal antibody was determined in anELISA procedure similar to the specificity tests described in Example Iexcept that the antigen plate contained a pool of PLL (poly-L-lysine)immobilized K. pneumoniae K8 and K11 serotypes. Positive reaction of the7E10 monoclonal antibody with the K. pneumoniae serotypes was observedonly with the anti-IgM reagent, demonstrating an IgM isotype for themonoclonal antibody. It will be appreciated by those skilled in the artthat if the process of this Example were repeated several times and theisotypes of intergenus cross-reactive monoclonal antibodies so obtainedwere determined, one would find additional isotypes, e.g., IgM and IgGisotypes.

3. In vitro functional activity of the 7E10 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement. The bacterial serotypes used hereinwere only inactivated in the presence of monoclonal antibody 7E10, anactive complement source, and human neutrophils (Table 8). When thisexperiment was repeated with serotypes unreactive with antibody 7E10, nobacterial destruction was observed (data not shown). These experimentsdemonstrated the functional specificity of monoclonal antibody 7E10, aswell as its capacity to opsonize bacteria and promote theirphagocytosis.

                  TABLE 8                                                         ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        S. marcescens                                                                          +        7E10      -.sup.a                                                                              0                                          012                                                                           S. marcescens                                                                          +        .sup. 6F11.sup.b                                                                       +       0                                          012                                                                           S. marcescens                                                                          -        7E10     +       0                                          012                                                                           S. marcescens                                                                          +        7E10     +       86%                                        012                                                                           K. pneumoniae                                                                          +        7E10     -       0                                          K8 and K11                                                                    K. pneumoniae                                                                          +        6F11     +       0                                          K8 and K11                                                                    K. pneumoniae                                                                          -        7E10     +       0                                          K8 and K11                                                                    K. pneumoniae                                                                          +        7E10     +       94%                                        K8 and K11                                                                    E. aerogenes                                                                           +        7E10     -       0                                          Isolates                                                                      E. aerogenes                                                                           +        6F11     +       0                                          Isolates                                                                      E. aerogenes                                                                           -        7E10     +       0                                          Isolates                                                                      E. aerogenes                                                                           +        7E10     +       60%                                        Isolates                                                                      ______________________________________                                         .sup.a and .sup.b = see Table 3 footnotes                                

EXAMPLE VI

EXAMPLE VI demonstrates methods for the production and selection of ahuman monoclonal antibody that possesses intergenus cross-reactivityagainst members of the genera Serratia marcescens and Klebsiellapneumoniae. Further, this example demonstrates the in vitro opsonicactivity of said antibody against homologous S. marcescens and K.pneumoniae serotypes. The process of Example I (essentially as describedin parts A through G) was repeated, except that it was necessary to makespecific modifications to characterize and assay the antibody describedin this Example. The following are changes in assay procedures and theresults obtained with the monoclonal antibody described herein.

1. Culture supernatants from four transformations were analyzed by theabove method resulting in the identification of one well (8C9) whichpossessed binding activity on at least one of four K. pneumoniaeserotype plates, containing the capsule serotypes: 1, 2, 3, 4, 6, 8, 9,19, 20 21, 24 27, 31, 43, 44, and 55, but not on the control plate (nobacteria). Antibody from the 8C9 cell line was assayed for furtherintergenus cross-reactivity by a modification of the standardimmunoblotting technique. Specifically, cross-reactivity to the bacteriaK. pneumoniae, E. aerogenes, S. marcescens, E. coli, and P. aeruginosawas investigated by spotting bacteria onto a gridded nitrocellulosepaper disc, reacting the disc containing the bacteria with saidantibody, and developing the antibody reactions with an alkalinephosphatase/nitroblue tetrazolium enzyme system.

From these experiments, the 8C9 antibody was observed to possess furtherintergenus cross-reactivity. This antibody reacted with the followingspecies and serotypes:

    ______________________________________                                        K. pneumoniae    S. marcescens                                                ______________________________________                                        K5,6,7,14,27,36,55,64                                                                          03                                                           ______________________________________                                    

Thus, the human monoclonal antibody 8C9 possessed intergenuscross-reactivity to bacteria belonging to the genera K. pneumoniae andS. marcescens.

2. The isotype of the 8C9 monoclonal antibody was determined in an ELISAprocedure similar to the specificity tests described in Example I exceptthat the antigen plate contained a pool of PLL immobilized K. pneumoniaeK14 and K27 serotypes. Positive reaction of the 8C9 monoclonal antibodywith the K. pneumoniae serotypes was observed only with the anti-IgMreagent, demonstrating an IgM isotype for the monoclonal antibody. Itwill be appreciated by those skilled in the art that if the process ofthis example were repeated several times and the isotypes of intergenuscross-reactive monoclonal antibodies so obtained were determined, onewould find additional isotypes, e.g., IgM and IgG isotypes.

3. In vitro functional activity of the 8C9 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement. The bacterial serotypes used hereinwere only inactivated in the presence of monoclonal antibody 8C9, anactive complement source, and human neutrophils (Table 9). When thisexperiment was repeated with serotypes unreactive with antibody 8C9, nobacterial destruction was observed (data not shown). These experimentsdemonstrated the functional specificity of monoclonal antibody 8C9, aswell as its capacity to opsonize bacteria and promote theirphagocytosis.

                  TABLE 9                                                         ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        S. marcescens                                                                          +        8C9       -.sup.a                                                                              0                                          03                                                                            S. marcescens                                                                          +        .sup. 6F11.sup.b                                                                       +       0                                          03                                                                            S. marcescens                                                                          -        8C9      +       0                                          03                                                                            S. marcescens                                                                          +        8C9      +       70%                                        03                                                                            K. pneumoniae                                                                          +        8C9      -       0                                          K14 and 27                                                                    K. pneumoniae                                                                          +        6F11     +       0                                          K14 and 27                                                                    K. pneumoniae                                                                          -        8C9      +       0                                          K14 and 27                                                                    K. pneumoniae                                                                          +        8C9      +       93%                                        K14 and 27                                                                    ______________________________________                                         .sup.a and .sup.b = see Table 3 footnotes                                

EXAMPLE VII

EXAMPLE VII demonstrates methods for the production and selection of ahuman monoclonal antibody that possesses intergenus cross-reactivityagainst members of the genera Serratia marcescens, Klebsiellapneumoniae, Enterobacter aerogenes, and Enterobacter cloacae. Further,this example demonstrates the in vitro opsonic activity of said antibodyagainst homologous S. marcescens, K. pneumoniae, E. aerogenes, and E.cloacae serotypes. The process of Example I (essentially as described inparts A through G) was repeated, except that it was necessary to makespecific modifications to characterize and assay the antibody describedin this Example. The following are changes in assay procedures and theresults obtained with the monoclonal antibody described herein.

1. Culture supernatants from four transformations were analyzed by theabove method resulting in the identification of one well (1E4) whichpossessed binding activity on at least one of four K. pneumoniaeserotype plates, containing the capsule serotypes: 1, 2, 3, 4, 6, 8, 9,19, 20, 21, 24 27, 31, 43, 44, and 55, but not on the control plate (nobacteria). Antibody from the 1E4 cell line was assayed for furtherintergenus cross-reactivity by a modification of the standardimmunoblotting technique. Specifically, cross-reactivity to the bacteriaK. pneumoniae, E. aerogenes, S. marcescens, E. coli, E. cloacae, and P.aeruginosa was investigated by spotting bacteria onto a griddednitrocellulose paper disc, reacting the disc containing the bacteriawith said antibody, and developing the antibody reactions with analkaline phosphatase/nitroblue tetrazolium enzyme system.

From these experiments, the 1E4 antibody was observed to possess furtherintergenus cross-reactivity. This antibody reacted with the followingspecies and serotypes:

    ______________________________________                                        K. pneumoniae                                                                            S. marcescens                                                                              E. aerogenes                                                                            E. cloacae                                  ______________________________________                                        K1,3,8,9,13,                                                                             015          Clinical  Clinical                                    15,29,31,33,36,         Isolates  Isolates                                    68,69                                                                         ______________________________________                                    

Thus, the human monoclonal antibody 1E4 possessed intergenuscross-reactivity to bacteria belonging to the species K. pneumoniae, S.marcescens, E. cloacae and E. aerogenes.

2. The isotype of the 1E4 monoclonal antibody was determined in an ELISAprocedure similar to the specificity tests described in Example I exceptthat the antigen plate contained a pool of PLL immobilized K. pneumoniaeK3 and K8 serotypes. Positive reaction of the 1E4 monoclonal antibodywith the K. pneumoniae serotypes was observed only with the anti-IgMreagent, demonstrating an IgM isotype for the monoclonal antibody. Itwill be appreciated by those skilled in the art that if the process ofthis example were repeated several times and the isotypes of intergenuscross-reactive monoclonal antibodies so obtained were determined, onewould find additional isotypes, e.g., IgM and IgG isotypes.

3. In vitro functional activity of the 1E4 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement. The bacterial serotypes used hereinwere only inactivated in the presence of monoclonal antibody 1E4, anactive complement source, and human neutrophils (Table 10). When thisexperiment was repeated with serotypes unreactive with antibody 1E4, nobacterial destruction was observed (data not shown). These experimentsdemonstrated the functional specificity of monoclonal antibody 1E4, aswell as its capacity to opsonize bacteria and promote theirphagocytosis.

                  TABLE 10                                                        ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        S. marcescens                                                                          +        1E4       -.sup.a                                                                              0                                          015                                                                           S. marcescens                                                                          +        .sup. 6F11.sup.b                                                                       +       0                                          015                                                                           S. marcescens                                                                          -        1E4      +       0                                          015                                                                           S. marcescens                                                                          +        1E4      +       86%                                        015                                                                           K. pneumoniae                                                                          +        1E4      -       0                                          K3 and 29                                                                     K. pneumoniae                                                                          +        6F11     +       0                                          K3 and 29                                                                     K. pneumoniae                                                                          -        1E4      +       0                                          K3 and 29                                                                     K. pneumoniae                                                                          +        1E4      +       80%                                        K3 and 29                                                                     E. aerogenes                                                                           +        1E4      -       0                                          Isolate (2)                                                                   E. aerogenes                                                                           +        6F11     +       0                                          Isolate (2)                                                                   E. aerogenes                                                                           -        1E4      +       0                                          Isolate (2)                                                                   E. aerogenes                                                                           +        1E4      +       80%                                        Isolate (2)                                                                   E. cloacae                                                                             +        1E4      -       0                                          Isolate                                                                       E. cloacae                                                                             +        6F11     +       0                                          Isolate                                                                       E. cloacae                                                                             -        1E4      +       0                                          Isolate                                                                       E. cloacae                                                                             +        1E4      +       80%                                        Isolate                                                                       ______________________________________                                         .sup.a and .sup.b = see Table 3 footnotes                                

EXAMPLE VIII

EXAMPLE VIII demonstrates methods for the production and selection of ahuman monoclonal antibody that possesses intergenus cross-reactivityagainst members of the genera Serratia marcescens, Klebsiellapneumoniae, Enterobacter aerogenes, and Pseudomonas aeruginosa. Further,this example demonstrates the in vitro opsonic activity of said antibodyagainst homologous S. marcescens, K. pneumoniae, E. aerogenes, and P.aeruginosa serotypes. The process of Example I (essentially as describedin parts A through G) was repeated, except that it was necessary to makespecific modifications to characterize and assay the antibody describedin this Example. The following are changes in assay procedures and theresults obtained with the monoclonal antibody described herein.

1. Culture supernatants from four transformations were analyzed by theabove method resulting in the identification of one well (9D1) whichpossessed binding activity on at least one of four K. pneumoniaeserotype plates, containing the capsule serotypes; 1, 2, 3, 4, 6, 8, 9,19, 20, 24, 27, 31, 43, 44, and 55, but not on the control plate (nobacteria). Antibody from the 9D1 cell line was assayed for furtherintergenus cross-reactivity by a modification of the standardimmunoblotting technique. Specifically, cross-reactivity to the bacteriaK. pneumoniae, E. aerogenes, S. marcescens, E. coli, and P. aeruginosawas investigated by spotting bacteria onto a gridded nitrocellulosepaper disc, reacting the disc containing the bacteria with saidantibody, and developing the antibody reactions with an alkalinephosphatase/nitroblue tetrazolium enzyme system.

From these experiments, the 9D1 antibody was observed to possess furtherintergenus cross-reactivity. This antibody reacted with the followingspecies and serotypes:

    ______________________________________                                        K. pneumoniae                                                                           S. marcescens                                                                             E. aerogenes                                                                             P. aeruginosa                                ______________________________________                                        E9,13,15,29,                                                                            03,9,15,18  Clinical   F6                                           33                    Isolates                                                ______________________________________                                    

Thus, the human monoclonal antibody 9D1 possessed intergenuscross-reactivity to bacteria belonging to the genera K. pneumoniae, S.marcescens, E. aerogenes, and P. aeruginosa.

2. The isotype of the 9D1 monoclonal antibody was determined in an ELISAprocedure similar to the specificity tests described in Example I exceptthat the antigen plate contained a pool of PLL immobilized K. pneumoniaeK13 serotype. Positive reaction of the 9D1 monoclonal antibody with theK. pneumoniae serotypes was observed only with the anti-IgM reagent,demonstrating an IgM isotype for the monoclonal antibody. It will beappreciated by those skilled in the art that if the process of thisexample were repeated several times and the isotypes of intergenuscross-reactive monoclonal antibodies so obtained were determined, onewould find additional isotypes, e.g., IgM and IgG isotypes.

3. In vitro functional activity of the 9D1 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement. The bacterial serotypes used hereinwere only inactivated in the presence of monoclonal antibody 9D1, anactive complement source, and human neutrophils (Table 11). When thisexperiment was repeated with serotypes unreactive with antibody 9D1, nobacterial destruction was observed (data not shown). These experimentsdemonstrated the functional specificity of monoclonal antibody 9D1, aswell as its capacity to opsonize bacteria and promote theirphagocytosis.

                  TABLE 11                                                        ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        S. marcescens                                                                          +        9D1       -.sup.a                                                                              0                                          03                                                                            S. marcescens                                                                          +        .sup. 6F11.sup.b                                                                       +       0                                          03                                                                            S. marcescens                                                                          -        9D1      +       0                                          03                                                                            S. marcescens                                                                          +        9D1      +       87%                                        03                                                                            K. pneumoniae                                                                          +        9D1      -       0                                          K13                                                                           K. pneumoniae                                                                          +        6F11     +       0                                          K13                                                                           K. pneumoniae                                                                          -        9D1      +       0                                          K13                                                                           K. pneumoniae                                                                          +        9D1      +       50%                                        K13                                                                           E. aerogenes                                                                           +        9D1      -       0                                          Isolates (2)                                                                  E. aerogenes                                                                           +        6F11     +       0                                          Isolates (2)                                                                  E. aerogenes                                                                           -        9D1      +       0                                          Isolates (2)                                                                  E. aerogenes                                                                           +        9D1      +       70%                                        Isolates (2)                                                                  P. aeruginosa                                                                          +        9D1      -       0                                          F6                                                                            P. aeruginosa                                                                          +        6F11     +       0                                          F6                                                                            P. aeruginosa                                                                          -        9D1      +       0                                          F6                                                                            P. aeruginosa                                                                          +        9D1      +       75%                                        F6                                                                            ______________________________________                                         .sup.a and .sup.b = see Table 3 footnotes                                

EXAMPLE IX

EXAMPLE IX demonstrates methods for production of a human monoclonalantibody that possesses intergenus cross-reactivity against members ofthe species Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E.coli), and Serratia marcescens (S. marcescens). Further, this Exampledemonstrates the in vivo protective activity of said antibody against alethal challenge of homologous P. aeruginosa, E. coli, and S. marcescensserotypes. The process of Example I (essentially as described in parts Athrough G) was repeated to produce a human monoclonal antibody that wascross-protective against infections caused by the bacteria to which itbinds. Specific modifications to the process of Example I, tocharacterize and assay the antibody, are described in this Example. Thechanges in assay procedures and the results obtained with the monoclonalantibody were as follows.

1. Supernatants were screened for the presence of anti-P. aeruginosaantibodies using an ELISA technique as described in Example I. Theantigen plate consisted of a flat-bottom 96-well Immunolon 2 microtiterplate (Dynatech, Alexandria, Va.), the wells of which contained amixture of poly-L-lysine (PLL) immobilized bacteria belonging to theseven P. aeruginosa Fisher reference strains (Fisher, M. W., et al., J.of Bacteriology (1969) 98:835-836, A.T.C.C. Nos. 27312-27318).

Culture supernatants from one transformation were analyzed by the abovemethod and resulted in the identification of one well that possessedactivity on the P. aeruginosa plate, but not the PLL control plate. Itwas determined in subsequent ELISA's with the seventeen individual P.aeruginosa serotypes belonging to the International Antigenic TypingScheme (IATS, A.T.C.C. Nos. 33348-33364), that one master well 9C3contained antibodies which bound to IATS serotype type 1 (Liu, P. V.,Int. J. Syst. Bacteriol. (1983) 33:256-264, which is incorporated hereinby reference).

Thus, from this experiment, one cloned transformed human cell line wasachieved which is continuous (immortal) and secretes a single humanmonoclonal antibody which binds to a determinant on the surface of theP. aeruginosa IATS type 1.

Prior to filing this patent application, the continuous transformedhuman cell line identified as 9C3 was deposited with the American TypeCulture Collection, Rockville, Md., at A.T.C.C. No. CRL 9239.

2. Antibody from the cloned 9C3 cell line was also assayed forcross-reactivity to gram-negative and gram-positive bacteria by amodification of the standard immunoblotting technique. Specifically,cross-reactivity to the bacteria E. coli, K. pneumoniae, S. marcescens,E. aerogenes, E. cloacae, Haemophilus influenzae, and Staphylococcusaureus was investigated by spotting bacteria onto a griddednitrocellulose paper disc, reacting the disc containing the bacteriawith 9C3 antibody, and visualizing the antibody reactions with analkaline phosphatase/nitroblue tetrazolium enzyme system (as describedin Example I).

From these experiments, antibody 9C3 was observed to bind to E. coliserotype 06 and the S. marcescens serotypes 012 and 014. Thus, the humanmono- clonal antibody 9C3 possesses intergenus cross-reactivity amongthe gram-negative bacteria belonging to specific serotypes of thespecies E. coli, S. marcescens, and P. aeruginosa.

3. The finding that the monoclonal antibody cross-reacted with severaldifferent bacterial genera, suggested the antibody may bind to a sharedantigenic determinant. The biochemical characterization of the molecularspecies recognized by the 9C3 antibody was accomplished by immunoblotanalysis as described in Example I. Reactions were noted in deoxycholateextracts of reactive serotypes from P. aeruginosa and E. coli, but notS. marcescens. Although it is not clear why antibody 9C3 was unreactivewith the S. marcescens preparation, it is possible the antibodyrecognizes a conformational epitope that was destroyed by thepreparation treatments. For the E. coli and P. aeruginosa bacterialextracts, the 9C3 antibody appeared to recognize a series of regularlyspaced molecular entities giving rise to a ladder-like pattern on theimmunoblot. This profile was entirely consistent with that seen inpolyacrylamide gel electrophoretic analysis of LPS in the presence ofSDS, where it has been demonstrated that the heterogeneous size profileexhibited by the bands is due to a population of LPS molecules differingby weight increments equivalent to the number of O-antigenicoligosaccharide side chain units present per molecule (Pavla, E. T., andMakela, P. H., supra, and Goldman, R. D., and Leive, L., supra).

To further define the molecular nature of the antigen, the deoxycholateextracts were treated with proteolytic enzyme, Proteinase K, prior totheir electrophoresis (Eberling, W., supra). The immunoblot patternsobserved after Proteinase K treatment were identical to those patternsobserved without treatment and, thus, suggested that the antigenreactive with the 9C3 antibody was not protein in nature.

4. The isotype of the 9C3 monoclonal antibody was determined in an ELISAprocedure similar to the specificity tests described above, except thatthe antigen plate contained PLL-immobilized P. aeruginosa Fisherserotype 4 bacteria. Positive reaction of the 9C3 monoclonal antibodywith the P. aeruginosa strain was observed only with the anti-IgMreagent, demonstrating an IgM isotype for the monoclonal antibody.

5. In vitro functional activity of the 9C3 monoclonal antibody wasexamined in an opsonophagocytic assay which compared the bacteriocidalactivity of the antibody in the presence and absence of both humanneutrophils and human complement.

The bacterial strains used herein were only killed in the presence onmonoclonal antibody 9C3, an active complement source, and humanneutrophils (Table 12). When this experiment was repeated with severalnon-9C3 reactive bacterial serotypes, no bacterial destruction wasobserved, thus demonstrating the functional specificity of monoclonalantibody 9C3 and its capacity to opsonize bacteria and promote theirphagocytosis. Since the combined action of opsonins (specificantibodies) and polymorphonuclear leukocytes (neutrophils) appeared tobe the primary mechanism for immunity to these bacterial strains, thesedata indicate that antibody 9C3 would, after appropriate administration,provide protection against lethal challenges with the bacteria strainsdescribed herein.

                  TABLE 12                                                        ______________________________________                                                 Neutro-                   % Destruction of                           Bacteria phils    Antibody Complement                                                                            Input Bacteria                             ______________________________________                                        E. coli 06                                                                             +        9C3        -.sup.(a)                                                                           0                                          E. coli 06                                                                             +          6F11.sup.(b)                                                                         +       0                                          E. coli 06                                                                             -        9C3      +       0                                          E. coli 06                                                                             +        9C3      +       98%                                        S. marcescens                                                                          +        9C3      -       0                                          014                                                                           S. marcescens                                                                          +        6F11     +       0                                          014                                                                           S. marcescens                                                                          -        9C3      +       0                                          014                                                                           S. marcescens                                                                          +        9C3      +       94%                                        014                                                                           P. aeruginosa                                                                          +        9C3      -       0                                          Fisher 4                                                                      P. aeruginosa                                                                          +        6F11     +       0                                          Fisher 4                                                                      P. aeruginosa                                                                          -        9C3      +       0                                          Fisher 4                                                                      P. aeruginosa                                                                          +        9C3      +       79%                                        Fisher 4                                                                      ______________________________________                                         .sup.(a) - = heatinactivated (56° C. for 30 min) human complement.     .sup.(b) 6F11 = culture supernatant containing an IgM human monoclonal        antibody to Pseudomonas aeruginosa Fisher type 2.                        

6. To test the protective characteristics of the 9C3 antibody, animalprotection studies were performed with at least one organism from eachgenus described herein.

The burned mouse model was used for protection experiments with P.aeruginosa Fisher 4 and S. marcescens 014. For each bacterial challenge,female, outbred Swiss-Webster mice weighing 22-25 gm were divided intothree groups of 7 or 8 mice each. An exemplary experiment was performedas follows:

    ______________________________________                                        Group        Bacteria    Antibody                                             ______________________________________                                        A            P. aeruginosa F4                                                                          9C3                                                  B            P. aeruginosa F4                                                                          6F11                                                 C            P. aeruginosa F2                                                                          9C3                                                  ______________________________________                                    

The day before the experiment, each mouse was shaved and treated with adepilatory agent to remove all hair on the back (burn site). On theexperiment day, each animal received 0.1 ml in each thigh of ananesthetizing saline solution containing 0.7 ml 0.85% NaCl, 0.2 mlxylazine (20 mg/ml) and 0.1 ml ketamine (100 mg/ml), such that thedosage per mouse was 20 mg/kg zylazine and 180 mg/kg ketamine. Theanesthetized mice received a 10% of total body surface area,full-thickness, third degree gas flame burn. Immediately after woundinfliction, the mice were injected sub-eschar with 0.5 ml of 4° C.antibody containing spent culture fluid pre-mixed with 5-10 LD₅₀ 's ofbacteria. The bacterial suspension had been prepared from a brothculture in logarithmic phase growth, from which the bacteria werecentrifuged, washed twice in PBS, and resuspended to the appropriateconcentration in PBS. Animals were observed for a period of ten days.Three to five days post-challenge, all animals in Group B (irrelevantantibody) and Group C (irrelevant organism) were dead. In contrast,those animals that had received the 9C3 (Group A) antibody were allalive and symptom-free (see Table 13).

For the E. coli 06 protection studies, healthy Swiss-Webster mice (20-22gm) were divided into three groups of ten mice each. Each groupreceiving antibody was injected intravenously with 200 μl of sterile PBScontaining 25 μg of purified antibody. Two hours later, all animals werechallenged intraperitoneally with 300 μl of live bacteria containing 3LD₅₀ 's of their respective bacterial strain (for results, see Table13).

                  TABLE 13                                                        ______________________________________                                                                     Survival/                                        Expt. Challenge Bacteria                                                                         Antibody  Challenge                                                                            % Survival                                ______________________________________                                        1     P. aeruginosa F4                                                                           9C3       6/7    86%                                             P. aeruginosa F4                                                                             6F11.sup.(a)                                                                          0/7    0                                               P. aeruginosa F2.sup.(b)                                                                   9C3       0/7    0                                         2     E. coli 06   9C3        6/10  60%                                             E. coli 06   6F11       0/10  0                                         3     S. marcescens 014                                                                          9C3       8/8    100%                                            S. marcescens 014                                                                          6F11      0/8    0                                         ______________________________________                                         .sup.(a) 6F11 antibody is specific to Pseudomonas aeruginosa Fisher           immunotype 2 and serves as negative control antibody.                         .sup.(b) P. aeruginosa F2 is not reactive with the 9C3 antibody and serve     as a nonspecific control organism.                                       

These data demonstrate that the human monoclonal antibody 9C3 is able toprotect mice from lethal challenges with bacteria belonging to threegram-negative genera. The intergenus cross-reactive human mono- clonalantibody 9C3 was able to afford protection with antibody containingculture supernatant or purified antibody against infection by organismsbelonging to the gram-negative genera E. coli, S. marcescens, and P.aeruginosa.

From the foregoing, it will be appreciated that the cell lines of thepresent invention provide compositions of human monoclonal antibodiesand fragments thereof cross-reactive for and cross-protective againstvarious bacterial species, both gram-negative and gram-positive. Thisallows prophylactic and therapeutic compositions to be more easilydeveloped that can be effective against nosocomial and neonatalinfections due to most, if not all, bacterial genera. By combining theantibodies, it is possible to obtain broad protection against a largeportion, usually less than all, of the clinically significant bacteria.In addition, the cell lines provide antibodies which find uses inimmunoassays and other well-known procedures.

The transformed human cell lines 9B10, 4F10, 4B9, and 7D7, describedherein, were deposited in the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, on Jan. 28, 1986, and given thedesignations CRL 9006, CRL 9007, CRL 9008, and CRL 9009, respectively.The transformed human cell line 9C3, described herein, was deposited inthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852, on Oct. 22, 1986, and given the designation CRL 9239.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

What is claimed is:
 1. A protective human monoclonal antibody or antigenbinding fragment thereof which specifically binds to an accessiblecarbohydrate determinant of outer membrane lipopolysaccharide thatimmunologically binds a monoclonal antibody produced by a cell linedesignated ATCC No. CRL 9006, CRL 9007, CRL 9009 or CRL
 9239. 2. Acomposition useful in the prophylactic or therapeutic treatment ofbacterial infection, said composition comprising a prophylactic ortherapeutic amount of at least two protective human monoclonalantibodies or antigen binding fragments thereof which individuallyimmunologically bind with an accessible carbohydrate determinant ofouter membrane lipopolysaccharide shared by serotypes of two or morebacterial species of different genera of Enterobacteriaceae and aplurality of serotypes in at least one of said species, wherein saidspecies comprise Escherichia coli, Serratia marcescens, Enterobacteraerogenes, and Enterobacter cloacae.
 3. A human monoclonal antibody orbinding fragment thereof useful in the treatment of bacterial infectionwhich immunologically binds with an accessible carbohydrate determinantof outer membrane lipopolysaccharide shared by serotypes of at least twodifferent bacterial species and which is protective against infection bybacteria of the serotypes, wherein one of the species is E. coli.
 4. Thehuman monoclonal antibody of claim 3, wherein the second species whichimmunologocally binds with the antibody is Enterobacter cloacae,Enterobacter aerogenes, Neisseria meningitidis, Serratia marcescens, orPseudomonas aeruginosa.
 5. The human monoclonal antibody of claim 4,which immunologically binds with E. coli, S. marcescens, and E.aerogenes.
 6. The monoclonal antibody of claim 5 which is obtained fromcell line ATCC CRL
 9009. 7. The human monoclonal antibody of claim 3,wherein the second and a subsequent species which immunologically bindthe antibody are S. marcescens and P. aeruginosa.
 8. The humanmonoclonal antibody of claim 7 which is obtained from cell line ATCC CRL9239.
 9. A human monoclonal antibody or binding fragment thereof usefulin the treatment of bacterial infection which immunologically binds anaccessible carbohydrate determinant of outer membrane lipopolysaccharideshared by serotypes of at least two different bacterial species andwhich is protective against infection by bacteria of the serotypes,wherein one of the species is from the genus Enterobacter.
 10. The humanmonoclonal antibody of claim 9, wherein the second speciesimmunologically binding with the antibody is E. coli, S. marcescens, orP. aeruginosa.
 11. A human monoclonal antibody of claim 10 whichimmunologically binds with E. aerogenes and S. marcescens.
 12. The humanmonoclonal antibody of claim 11 which is obtained from cell line ATCCCRL
 9007. 13. A pharmaceutical composition for the treatment of abacterial infection in a host which comprises a human monoclonalantibody of claims 3 or 9 and a physiologically acceptable carrier. 14.A human monoclonal antibody useful in the treatment of bacterialinfection, having the antigen binding specificity of the antibodyproduced by the cell line ATCC No. CRL
 9009. 15. A pharmaceuticalcomposition for the treatment of bacterial infection in a host whichcomprises the human monoclonal antibody of claim 14 and aphysiologically acceptable carrier.
 16. A human monoclonal antibodyuseful in the treatment of bacterial infection, having the antigenbinding specificity of the antibody produced by the cell line ATCC No.CRL
 9239. 17. A pharmaceutical composition for the treatment ofbacterial infection in a host which comprises the human monoclonalantibody of claim 16 and a physiologically acceptable carrier.
 18. Ahuman monoclonal antibody useful in the treatment of bacterialinfection, the antibody produced by the cell line ATCC No. CRL
 9006. 19.A pharmaceutical composition for the treatment of bacterial infection ina host which comprises the human monoclonal antibody of claim 18 and aphysiologically acceptable carrier.
 20. A human monoclonal antibodyuseful in the treatment of bacterial infection, having the antigenbinding specificity of the antibody produced by the cell line ATCC No.CRL
 9007. 21. A pharmaceutical composition for the treatment ofbacterial infection in a host which comprises the human monoclonalantibody of claim 20 and a physiologically acceptable carrier.
 22. Thehuman monoclonal antibody of claim 14 which is produced by the cell lineATCC No. CRL
 9009. 23. The human monoclonal antibody of claim 16 whichis produced by the cell line ATCC No. CRL 9239.